IP-10/MIG receptor designated CXCR3, nucleic acids, and methods of use therefor

ABSTRACT

The invention relates to isolated and/or recombinant nucleic acids encoding a mammalian (e.g., human) CXCR3 protein and variants thereof, including antisense nucleic acid, recombinant nucleic acid constructs, such as plasmids or retroviral vectors, comprising a nucleic acid which encodes a protein of the present invention or variant thereof, and to host cells comprising a nucleic acid or construct, useful in the production of recombinant proteins. The invention also relates to a method for producing a mammalian (e.g., human) CXCR3 protein.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.08/709,838, now U.S. Pat. No. 6,140,064 filed Sep. 10, 1996.

BACKGROUND

Chemokines constitute a family of small cytokines that are produced ininflammation and regulate leukocyte recruitment (Baggiolini, M. et al.,“Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines,”Adv. Immunol. 55: 97-179 (1994); Springer, T. A., “Traffic signals onendothelium for lymphocyte recirculation and leukocyte emigration,”Annu. Rev. Physiol. 57: 827-872 (1995); and Schall, T. J. and K. B.Bacon, “Chemokines, leukocyte trafficking, and inflammation,” Curr.Opin. Immunol. 6: 865-873 (1994)). Chemokines are capable of selectivelyinducing chemotaxis of the formed elements of the blood (other than redblood cells), including leukocytes such as neutrophils, monocytes,macrophages, eosinophils, basophils, mast cells, and lymphocytes, suchas T cells and B cells. In addition to stimulating chemotaxis, otherchanges can be selectively induced by chemokines in responsive cells,including changes in cell shape, transient rises in the concentration ofintracellular free calcium ions ([Ca²⁺]_(i)), granule exocytosis,integrin upregulation, formation of bioactive lipids (e.g.,leukotrienes) and respiratory burst, associated with leukocyteactivation. Thus, the chemokines are early triggers of the inflammatoryresponse, causing inflammatory mediator release, chemotaxis andextravasation to sites of infection or inflammation.

Two subfamilies of chemokines, designated as CXC and CC chemokines, aredistinguished by the arrangement of the first two of four conservedcysteine residues, which are either separated by one amino acid (as inCXC chemokines IL-8, γIP-10, Mig, PF4, ENA-78, GCP-2, GROα, GROβ, GROγ,NAP-2, NAP-4) or are adjacent residues (as in CC chemokines MIP-1α,MIP-1β, RANTES, MCP-1, MCP-2, MCP-3, I-309). Most CXC chemokines attractneutrophil leukocytes. For example, the CXC chemokines interleukin 8(IL-8), platelet factor 4 (PF4), and neutrophil-activating peptide 2(NAP-2) are potent chemoattractants and activators of neutrophils. TheCXC chemokines designated Mig (monokine induced by gamma interferon) andIP-10 (γIP-10, interferon-gamma inducible 10 kDa protein) areparticularly active in inducing chemotaxis of activated peripheral bloodleukocytes. CC chemokines are generally less selective and can attract avariety of leukocyte cell types, including monocytes, eosinophils,basophils, T lymphocytes and natural killer cells. CC chemokines such ashuman monocyte chemotactic proteins 1-3 (MCP-1, MCP-2 and MCP-3), RANTES(Regulated on Activation, Normal T Expressed and Secreted), and themacrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) have beencharacterized as chemoattractants and activators of monocytes orlymphocytes, but do not appear to be chemoattractants for neutrophils.

Chemokines act through receptors which belong to a superfamily of seventransmembrane spanning G-protein coupled receptors (Murphy, P. M., “Themolecular biology of leukocyte chemoattractant receptors,” Annu. Rev.Immunol., 12: 593-633 (1994); Gerard, C. and N. P. Gerard, “Thepro-inflammatory seven transmembrane segment receptors of theleukocyte,” Curr. Opin. Immunol., 6: 140-145 (1994)). This family ofG-protein coupled (serpentine) receptors comprises a large group ofintegral membrane proteins, containing seven transmembrane-spanningregions. The receptors are coupled to G proteins, which areheterotrimeric regulatory proteins capable of binding GTP and mediatingsignal transduction from coupled receptors, for example, by theproduction of intracellular mediators. Two of these receptors, theinterleukin-8 (IL-8) receptors, IL-8R1 (interleukin-8 receptor type 1;Holmes, W. E. et al., “Structure and functional expression of a humaninterleukin-8 receptor,” Science, 253: 1278-1280 (1991)) and IL-8R2(interleukin-8 receptor type 1; Murphy, P. M. and H. L. Tiffany,“Cloning of complementary DNA encoding a functional human interleukin-8receptor,” Science, 253: 1280-1283 (1991)), are largely restricted toneutrophils and recognize the NH2-terminal Glu-Leu-Arg (ELR) motif, anessential binding epitope in those CXC chemokines that induce neutrophilchemotaxis (Clark-Lewis, I. et al., “Structure-activity relationships ofinterleukin-8 determined using chemically synthesized analogs. Criticalrole of NH2-terminal residues and evidence for uncoupling of neutrophilchemotaxis, exocytosis, and receptor binding activities,” J. Biol.Chem., 266: 23128-23134 (1991); Hébert, C. A. et al., “Scanningmutagenesis of interleukin-8 identifies a cluster of residues requiredfor receptor binding,” J. Biol. Chem., 266: 18989-18994 (1991); andClark-Lewis, I. et al., “Platelet factor 4 binds to interleukin 8receptors and activates neutrophils when its N terminus is modified withGlu-Leu-Arg,” Proc. Natl. Acad. Sci. USA, 90: 3574-3577 (1993)). Fivedistinct CC chemokine receptors have been described, and are designatedCC-CKR1, -2, -3, -4 and -5 (CC-CKR, CC chemokine receptor; Neote, K. etal., “Molecular cloning, functional expression, and signalingcharacteristics of a CC chemokine receptor,” Cell, 72: 415-425 (1993);Gao, J.-L. et al., “Structure and functional expression of the humanmacrophage inflammatory protein 1α/RANTES receptor,” J. Exp. Med., 177:1421-1427 (1993); Charo, I. F. et al., “Molecular cloning and functionalexpression of two monocyte chemoattractant protein 1 receptors revealsalternative splicing of the carboxyl-terminal tails,” Proc. Natl. Acad.Sci. USA, 91: 2752-2756 (1994); Myers, S. J., et al., J. Biol. Chem.,270: 5786-5792 (1995); Combadiere, C. et al., Cloning and functionalexpression of a human eosinophil CC chemokine receptor,” J. Biol. Chem.,270 (27): 16491-16494 (1995); and Correction, J. Biol. Chem., 270: 30235(1995); Ponath, P. D. et al., “Molecular cloning and characterization ofa human eotaxin receptor expressed selectively on eosinophils,” J. Exp.Med., 183: 2437-2448 (1996); and Daugherty, B. L. et al., “Cloning,expression, and characterization of the human eosinophil eotaxinreceptor,” J. Exp. Med., 183: 2349-2354 (1996); Power, C. A. et al.,1995, “Molecular cloning and functional expression of a novel CCchemokine receptor cDNA from a human basophilic cell line,” J. Biol.Chem., 270: 19495-19500 (1995); Hoogewerf, A. J. et al.,” Molecularcloning of murine CC CKR-4 and high affinity binding of chemokines tomurine and human CC CKR-4,” Biochem. Biophys. Res. Commun., 218: 337-343(1996); Samson, M. et al., “Molecular cloning and functional expressionof a new human CC-chemokine receptor gene,” Biochemistry, 35: 3362-3367(1996)). The CC chemokine receptors occur on several types ofleukocytes, including monocytes, granulocytes and lymphocytes, andrecognize CC chemokines, but not CXC chemokines.

In contrast to monocytes and granulocytes, lymphocyte responses tochemokines are not well understood. Notably, none of the receptors ofknown specificity appear to be restricted to lymphocytes and thechemokines that recognize these receptors cannot, therefore, account forevents such as the selective recruitment of T lymphocytes that isobserved in T cell-mediated inflammatory conditions. Moreover, althougha number of proteins with significant sequence similarity and similartissue and leukocyte subpopulation distribution to known chemokinereceptors have been identified and cloned, the ligands for thesereceptors remain undefined. Thus, these proteins are referred to asorphan receptors. The characterization of the ligand(s) of a receptor,is essential to an understanding of the interaction of chemokines withtheir target cells, the events stimulated by this interaction, includingchemotaxis and cellular activation of leukocytes, and the development oftherapies based upon modulation of receptor function.

SUMMARY OF THE INVENTION

The present invention relates to proteins or polypeptides, referred toherein as isolated and/or recombinant mammalian (e.g., a primate such asa human) IP-10/Mig receptor proteins designated CXC Chemokine Receptor 3(CXCR3) and variants thereof. Recombinant CXCR3 proteins and variantscan be produced in host cells as described herein. In one embodiment, aCXCR3 protein or variant thereof is characterized by selective binding(e.g., high affinity binding) of one or more chemokines, such as IP-10and/or Mig, and/or the ability to induce a (one or more) cellularresponse(s) (e.g., chemotaxis, exocytosis, release of one or moreinflammatory mediators).

Another aspect of the present invention relates to isolated and/orrecombinant nucleic acids which encode a mammalian (e.g., a primate suchas a human) CXCR3 protein or variant thereof. The invention furtherrelates to recombinant nucleic acid constructs, such as plasmids orretroviral vectors, which contain a nucleic acid which encodes a proteinof the present invention or a variant thereof. The nucleic acids andconstructs can be used to produce recombinant receptor proteins and hostcells comprising a construct. In another embodiment, the nucleic acidencodes an antisense nucleic acid which can hybridize with a secondnucleic acid encoding a CXCR3 protein and which, when introduced intocells, can inhibit the expression of receptor.

Antibodies reactive with CXCR3 receptors can be produced using theproteins or variants thereof (e.g., a peptide) or cells expressingreceptor protein or variant as immunogen, for example. Such antibodiesor fragments thereof are useful in therapeutic, diagnostic and researchapplications, including the purification and study of the receptorproteins, identification of cells expressing surface receptor, andsorting or counting of cells.

Also encompassed by the present invention are methods of identifyingligands of the receptor, inhibitors (e.g., antagonists) or promoters(e.g., agonists) of receptor function. In one embodiment, suitable hostcells which have been engineered to express a receptor protein orvariant encoded by a nucleic acid introduced into said cells are used inan assay to identify and assess the efficacy of ligands, inhibitors orpromoters of receptor function. Such cells are also useful in assessingthe function of the expressed receptor protein or polypeptide.

According to the present invention, ligands, inhibitors and promoters ofreceptor function can be identified in a suitable assay, and furtherassessed for therapeutic effect. Inhibitors of receptor function can beused to inhibit (reduce or prevent) receptor activity, and ligandsand/or promoters can be used to induce (trigger or enhance) normalreceptor function where indicated. Thus, the present invention providesa method of treating inflammatory diseases including autoimmune diseaseand graft rejection, comprising administering an inhibitor of receptorfunction to an individual (e.g., a mammal). The present inventionfurther provides a method of stimulating receptor function byadministering a ligand or promoter to an individual, providing a newapproach to selective stimulation of leukocyte function, which isuseful, for example, in the treatment of infectious diseases and cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the nucleotide sequence determined from the1670 bp insert of a cDNA encoding a human IP-10/Mig receptor, which wasisolated from a human CD4⁺ T cell (KT30) cDNA library (SEQ ID NO:1). Anopen reading frame (69-1175) encodes a predicted protein of 368 aminoacids (SEQ ID NO:2). A putative poly-A signal and poly A site arelocated at positions 1534-1539 and at 1624-1670, respectively.

FIG. 2 is an illustration of the conceptual translation of the openreading frame of the sequence in FIG. 1, which encodes a human IP-10/Migreceptor (SEQ ID NO:2). Arrowheads indicate potential N-linkedglycosylation sites and horizontal lines indicate the location ofputative transmembrane domains (TM1-TM7).

FIGS. 3A-3C are graphs illustrating the responses induced by IP-10 andMig in stably transfected cells expressing IP-10/MigR. FIG. 3A is agraph illustrating the concentration dependent [Ca²⁺]_(i) changes inIP-10/MigR-transfected 300-19 cells. IP-10 or Mig were each added at 1,10, and 100 nM to Fura-2/AM loaded cells (arrowhead), and[Ca²⁺]_(i)-dependent fluorescence changes were recorded. Non-transfectedcells (lower tracings) were stimulated with IP-10 or Mig at 100 nM underidentical conditions.

FIG. 3B is a graph illustrating the results of studies assessingreceptor desensitization and cross-desensitization, in which IP-10/MigRexpressing 300-19 cells were sequentially stimulated with 100 nM IP-10or Mig, and with IP-10 followed by Mig or vice versa, and fluorescencechanges were recorded.

FIG. 3C is a graph illustrating the chemotaxis of IP-10/MigR expressingJurkat cells stimulated with IP-10 (filled circles) or Mig (filledsquares). The lower panel shows the response of non-transfected Jurkatcells when stimulated with increasing amounts of IP-10 (open circles) orMig (open squares). Mean numbers (±SD) of migrating cells per five highpower fields are presented.

FIGS. 4A-4B are graphs illustrating the responses of peripheral bloodlymphocytes (PBL) to IP-10 and Mig. Freshly isolated PBL from donorblood buffy coats were used as such (lower tracings and open symbols),or were used after culturing for 10 days in the presence of IL-2 (400U/ml) (upper tracings and closed symbols). FIG. 4A is a graphillustrating [Ca²⁺]_(i) changes induced by IP-10 or Mig. IP-10 or Migwere each added at 1, 10, and 100 nM to Fura-2/AM loaded cultured cells(arrowhead), and [Ca²⁺]_(i)-dependent fluorescence changes were recorded(upper tracings). Freshly isolated cells (lower tracings) werestimulated with IP-10 or Mig at 100 nM under the same conditions.

FIG. 4B is a graph illustrating chemotaxis of PBL in response toincreasing concentrations of IP-10 (filled circles) or Mig (filledsquares) (mean numbers (±SD) of migrating cells per five high. powerfields are presented).

DETAILED DESCRIPTION OF THE INVENTION

As described herein, a nucleic acid encoding a novel chemokine receptorthat is selective for the CXC chemokines IP-10 and Mig was cloned andcharacterized. The clone, which was isolated from a human CD4⁺ T celllibrary, was not detected in monocyte- or granulocyte-derived cDNAlibraries. Sequence analysis of the clone revealed an open reading frameof 1104 base pairs (FIG. 1, SEQ ID NO:1), encoding a predicted proteinof 368 amino acids with a predicted molecular mass of 40,659 daltons(FIG. 2, SEQ ID NO:2). The amino acid sequence includes seven putativetransmembrane segments which are characteristic of G-protein coupledreceptors and are found in other chemoattractant receptors. Consistentwith this observation, the receptor mediates Ca²⁺ (calcium ion)mobilization and chemotaxis in response to IP-10 and Mig (Example 2). Nosignificant response to the CXC chemokines IL-8, GROα, NAP-2(neutrophil-activating protein-2), GCP-2 (granulocyte chemotacticprotein-2), ENA78 (epithelial-derived neutrophil-activating peptide 78),PF4 (platelet factor 4), or the CC chemokines MCP-1 (monocytechemotactic protein-1), MCP-2, MCP-3, MCP-4, MIP-1α (macrophageinflammatory protein-1α), MIP-1β, RANTES (regulated on activation,normal T cell expressed and secreted), I309, eotaxin or lymphotactin wasdetected under similar conditions.

The restricted expression of human CXCR3 in activated T lymphocytes andthe ligand selectivity of the receptor for IP-10 and Mig are noteworthy.The human receptor is highly expressed in IL-2 activated T lymphocytes,but was not detected in resting T lymphocytes, B lymphocytes, monocytesor granulocytes under the conditions used (Example 2). The selectiveexpression in activated T lymphocytes is of interest, because otherreceptors for chemokines which have been reported to attract lymphocytes(e.g., MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β and RANTES) are also found ingranulocytes, such as neutrophils, eosinophils and basophils, as well asmonocytes. These results suggest that the IP-10/Mig receptor designatedCXCR3 is involved in the selective recruitment of effector T cells.

The receptor recognizes two unusual CXC chemokines, designated IP-10 andMig. Although IP-10 and Mig both belong to the CXC subfamily, incontrast to IL-8 and other CXC chemokines which are potentchemoattractants for neutrophils, the primary targets of IP-10 and Migare lymphocytes, particularly effector cells such as activated orstimulated T lymphocytes and natural killer (NK) cells (Taub, D. D. etal., J. Exp. Med., 177: 18090-1814 (1993); Taub, D. D. et al., J.Immunol., 155: 3877-3888 (1995)). (NK cells are large granularlymphocytes, which lack a specific T cell receptor for antigenrecognition, but possess cytolytic activity against cells such as tumorcells and virally infected cells.) Consistently, IP-10 and Mig lack theELR motif, an essential binding epitope in those CXC chemokines thatefficiently induce neutrophil chemotaxis (Clark-Lewis, I. et al., J.Biol. Chem., 266: 23128-23134 (1991); Hébert, C. A. et al., J. Biol.Chem., 266: 18989-18994 (1991); and Clark-Lewis, I. et al., Proc. Natl.Acad. Sci. USA, 90: 3574-3577 (1993)). In addition, both recombinanthuman Mig and recombinant human IP-10 have been reported to inducecalcium flux in tumor infiltrating lymphocytes (TIL) (Liao, F. et al.,J. Exp. Med., 182: 1301-1314 (1995)). While IP-10 has been reported toinduce chemotaxis of monocytes in vitro (Taub, D. D. et al., J. Exp.Med.; 177: 1809-1814 (1993), the receptor responsible has not beenidentified), human Mig appears highly selective, and does not show suchan effect (Liao, F. et al., J. Exp. Med., 182: 1301-1314 (1995)). IP-10expression is induced in a variety of tissues in inflammatory conditionssuch as psoriasis, fixed DRUG eruptions, cutaneous delayed-typehypersensitivity responses, tuberculoid leprosy, and in experimentalglomerulonephritis, and experimental allergic encephalomyelitis. IP-10also has a potent in vivo antitumor effect that is T cell dependent, isreported to be an inhibitor of angiogenesis in vivo, and can inducechemotaxis and degranulation of NK cells in vitro, suggesting a role asa mediator of NK cell recruitment and degranulation (in tumor celldestruction, for example) (Luster, A. D. and P. Leder, J. Exp. Med.,178: 1057-1065 (1993); Luster, A. D. et al., J. Exp. Med. 182: 219-231(1995); Angiolillo, A. L. et al., J. Exp. Med., 182: 155-162 (1995);Taub, D. D. et al., J. Immunol., 155: 3877-3888 (1995)).

The expression patterns of IP-10 and Mig are also distinct in thatexpression of each is induced by interferon-gamma (IFNγ), while theexpression of IL-8 is down-regulated by IFNγ (Luster, A. D. et al.,Nature, 315: 672-676 (1985); Farber, J. M., Proc. Natl. Acad. Sci. USA,87: 5238-5242 (1990); Farber, J. M., Biochem. Biophys. Res. Commun., 192(1): 223-230 (1993), Liao, F. et al., J. Exp. Med., 182: 1301-1314(1995); Seitz, M. et al., “Enhanced production of neutrophil-activatingpeptide-1/interleukin-8 in rheumatoid arthritis,” J. Clin. Invest., 87:463-469 (1991); Galy, A. H. M. and H. Spits, “IL-1, IL-4, and IFN-gammadifferentially regulate cytokine production and cell surface moleculeexpression in cultured human thymic epithelial cells,” J. Immunol., 147:3823-3830 (1991)).

Chemokines have been recently recognized as the long-sought mediatorsfor the recruitment of lymphocytes. Several CC chemokines were found toelicit lymphocyte chemotaxis (Loetscher, P. et al., “The monocytechemotactic proteins, MCP-1, MCP-2 and MCP-3, are major attractants forhuman CD4⁺ and CD8⁺ T lymphocytes,” FASEB J., 8: 1055-1060 (1994)), butthey are also active on granulocytes and monocytes (Uguccioni, M. etal., “Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES,MIP-1α and MIP-1β on human monocytes,” Eur. J. Immunol., 25: 64-68(1995); Baggiolini, M. and C. A. Dahinden, “CC chemokines in allergicinflammation,” Immunol. Today, 15: 127-133 (1994)). The situation isdifferent for IP-10 and Mig, which are selective in their action onlymphocytes, including activated T lymphocytes and NK cells, and whichbind CXCR3, a receptor which does not recognize numerous otherchemokines and which displays a selective pattern of expression (Example2).

In view of these observations, it is reasonable to conclude that theformation of the characteristic infiltrates in delayed-typehypersensitivity lesions, sites of viral infection, and certain tumorsis a process mediated by via CXCR3 and regulated by CXCR3 expression.Lymphocytes, particularly T lymphocytes, bearing a CXCR3 receptor as aresult of activation can be recruited into inflammatory lesions, sitesof infection, or tumors by IP-10 and/or Mig, which can be inducedlocally by interferon-gamma. Thus, CXCR3 plays a role in the selectiverecruitment of lymphocytes, particularly effector cells such asactivated or stimulated T lymphocytes.

Proteins and Peptides

The present invention relates to isolated and/or recombinant (including,e.g., essentially pure) proteins or polypeptides designated mammalianCXCR3 proteins and variants thereof. In a preferred embodiment, theisolated and/or recombinant proteins of the present invention have atleast one property, activity or function characteristic of a mammalianCXCR3 protein (as defined herein), such as a binding activity (e.g.,ligand, inhibitor and/or promoter binding), a signalling activity (e.g.,activation of a mammalian G protein, induction of rapid and transientincrease in the concentration of cytosolic free calcium [Ca²⁺]_(i)),cellular response function (e.g., stimulation of chemotaxis, exocytosisor inflammatory mediator release by leukocytes), and/or an immunologicalproperty as defined herein. For example, some proteins of the presentinvention can selectively bind to IP-10 and/or Mig, mediate cellularsignalling and/or a response thereto in vitro and/or in vivo (e.g.,calcium flux, chemotaxis and/or degranulation especially of activated Tlymphocytes). For example, as shown herein, a human CXCR3 protein,produced in mammalian cells by expression of a cDNA clone, canselectively bind to CXC chemokines IP-10 and/or Mig, and mediatesignalling, and a cellular response (e.g., chemotaxis). In oneembodiment, proteins of the present invention can bind a CXC chemokinefrom the same or a different mammalian species (e.g., human IP-10,murine IP-10, human Mig, murine Mig) (human IP-10, Luster, A. D. et al.,Nature, 315: 672-676 (1985); murine IP-10 (also referred to as CRG-2),Vanguri, P. and J. M. Farber, J. Biol. Chem., 265: 15049 (1990) andLuster, A. D. and P. Leder, J. Exp. Med., 178: 1057-1065 (1993); murineMig, Farber, J. M., Proc. Natl. Acad. Sci. USA, 87: 5238-5242 (1990);human Mig, Farber, J. M., Biochem. Biophys. Res. Commun., 192 (1):223-230 (1993) and Liao, F. et al., J. Exp. Med., 182: 1301-1314(1995)).

Proteins or polypeptides referred to herein as “isolated” are proteinsor polypeptides purified to a state beyond that in which they exist inmammalian cells, and include proteins or polypeptides obtained bymethods described herein, similar methods or other suitable methods,including essentially pure proteins or polypeptides, proteins orpolypeptides produced by chemical synthesis (e.g., synthetic peptides),or by combinations of biological and chemical methods, and recombinantproteins or polypeptides which are isolated. The proteins can beobtained in an isolated state of at least about 50% by weight,preferably at least about 75% by weight, or in essentially pure form.Proteins or polypeptides referred to herein as “recombinant” areproteins or polypeptides produced by the expression of recombinantnucleic acids.

As used herein “mammalian CXCR3 protein” refers to naturally occurringor endogenous mammalian CXCR3 proteins and to proteins having an aminoacid sequence which is the same as that of a naturally occurring orendogenous corresponding mammalian CXCR3 protein (e.g., recombinantproteins). Accordingly, as defined herein, the term includes mammalianCXCR3 protein, including mature protein, polymorphic or allelicvariants, and other isoforms of mammalian CXCR3 (e.g., produced byalternative splicing or other cellular processes), and modified orunmodified forms of the foregoing (e.g., glycosylated, unglycosylated,phosphorylated or unphosphorylated CXCR3 proteins). Naturally occurringor endogenous mammalian CXCR3 proteins include wild type proteins suchas mature CXCR3, polymorphic or allelic variants and other isoformswhich occur naturally in mammals (e.g., humans, non-human primates).Such proteins can be recovered from a source which naturally producesmammalian CXCR3, for example. These proteins and mammalian CXCR3proteins having the same amino acid sequence as a naturally occurring orendogenous corresponding mammalian CXCR3, are referred to by the name ofthe corresponding mammal. For example, where the corresponding mammal isa human, the protein is designated as a human CXCR3 protein (e.g., arecombinant human CXCR3 produced in a suitable host cell).

“Functional variants” of mammalian CXCR3 proteins include functionalfragments, functional mutant proteins, and/or functional fusion proteins(e.g., produced via mutagenesis and/or recombinant techniques).Generally, fragments or portions of mammalian CXCR3 proteins encompassedby the present invention include those having a deletion (i.e., one ormore deletions) of an amino acid (i.e., one or more amino acids)relative to the mature mammalian CXCR3 protein (such as N-terminal,C-terminal or internal deletions). Fragments or portions in which onlycontiguous amino acids have been deleted or in which non-contiguousamino acids have been deleted relative to mature mammalian CXCR3 proteinare also envisioned.

Generally, mutants or derivatives of mammalian CXCR3 proteins,encompassed by the present invention include natural or artificialvariants differing by the addition, deletion and/or substitution of oneor more contiguous or non-contiguous amino acid residues, or modifiedpolypeptides in which one or more residues is modified, and mutantscomprising one or more modified residues. Preferred mutants are naturalor artificial variants of mammalian CXCR3 proteins differing by theaddition, deletion and/or substitution of one or more contiguous ornon-contiguous amino acid residues. Such mutations can be in a conservedregion or nonconserved region (compared to other CXC and/or CC chemokinereceptors), extracellular, cytoplasmic, or transmembrane region, forexample.

A “functional fragment or portion”, “functional mutant” and/or“functional fusion protein” of a mammalian CXCR3 protein refers to anisolated and/or recombinant protein or oligopeptide which has at leastone property, activity or function characteristic of a mammalian CXCR3receptor (as defined herein), such as a binding activity (e.g., ligand,inhibitor and/or promoter binding), a signalling activity (e.g.,activation of a mammalian G protein, induction of rapid and transientincrease in the concentration of cytosolic free calcium [Ca²⁺]_(i)),cellular response function (e.g., stimulation of chemotaxis, exocytosisor inflammatory mediator release by leukocytes), and/or an immunologicalproperty as defined herein.

As used herein, a protein or polypeptide having “at least oneimmunological property” of a mammalian CXCR3 protein is one which (a) isbound by at least one antibody of a selected epitopic specificity whichbinds to a naturally occurring or endogenous mammalian CXCR3 protein orto a protein having the same amino acid seqence as the naturallyoccurring or endogenous mammalian CXCR3 protein (e.g., human CXCR3),and/or (b) is an immunogen capable of inducing the formation in asuitable animal of an antibody of a selected epitopic specificity whichbinds to a naturally occurring or endogenous mammalian CXCR3 or to aprotein having the same amino acid seqence as the naturally occurring orendogenous mammalian CXCR3. For example, a suitable fragment cancross-react with an antibody which is raised against and/or reactivewith isolated mammalian CXCR3.

Suitable fragments or mutants can be identified by screening. Forexample, the N-terminal, C-terminal, or internal regions of the proteincan be deleted in a step-wise fashion and the resulting protein orpolypeptide can be screened using a suitable assay, such as an assaydescribed herein (e.g., chemotaxis, calcium flux). Where the resultingprotein displays activity in the assay, the resulting protein(“fragment”) is functional. Information regarding the structure andfunction of mammalian G protein coupled receptors, including CXCchemokine and CC chemokine receptors, provides a basis for dividingmammalian CXCR3 proteins into functional domains (Murphy, P. M., “Themolecular biology of leukocyte chemoattractant receptors,” Annu. Rev.Immunol., 12: 593-633 (1994) and Gerard, C. and N. P. Gerard, “Thepro-inflammatory seven transmembrane segment receptors of theleukocyte,” Curr. Opin. Immunol., 6: 140-145 (1994), and referencescited therein).

The term variant also encompasses fusion proteins, comprising amammalian CXCR3 proteins (e.g., human CXCR3) as a first moiety, linkedto a second moiety not occurring in the mammalian CXCR3 as found innature. Thus, the second moiety can be an amino acid, oligopeptide orpolypeptide. The first moiety can be in an N-terminal location,C-terminal location or internal to the fusion protein. In oneembodiment, the fusion protein comprises an affinity ligand (e.g., anenzyme, an antigen, epitope tag) as the first moiety, and a secondmoiety comprising a linker sequence and human CXCR3 or portion thereof.

Examples of mammalian CXCR3 proteins include proteins encoded by anucleic acid of the present invention, such as a protein having an aminoacid sequence as set forth or substantially as set forth in FIG. 2 (SEQID NO:2). In a preferred embodiment, a mammalian CXCR3 or variant (e.g.,a variant including the extracellular N-terminal segment) has an aminoacid sequence which is at least about 50% identical, more preferably atleast about 70% identical, and still more preferably at least about 80%identical, to the protein shown in FIG. 2 (SEQ ID NO:2).

It will be appreciated that isolated and/or recombinant mammalian CXCR3proteins and variants thereof can be modified, for example, byincorporation of or attachment (directly or indirectly (e.g., via alinker)) of a detectable label such as a radioisotope, spin label,antigen (e.g., epitope label such as a FLAG tag) or enzyme label,flourescent or chemiluminesent group and the like, and such modifiedforms are included within the scope of the invention.

Nucleic Acids, Constructs and Vectors

The present invention relates to isolated and/or recombinant (including,e.g., essentially pure) nucleic acids having sequences which encode amammalian (e.g., human) CXCR3 protein or variant thereof as describedherein. Nucleic acids referred to herein as “isolated” are nucleic acidsseparated away from the nucleic acids of the genomic DNA or cellular RNAof their source of origin (e.g., as it exists in cells or in a mixtureof nucleic acids such as a library), and may have undergone furtherprocessing. “Isolated” nucleic acids include nucleic acids obtained bymethods described herein, similar methods or other suitable methods,including essentially pure nucleic acids, nucleic acids produced bychemical synthesis, by combinations of biological and chemical methods,and recombinant nucleic acids which are isolated. Nucleic acids referredto herein as “recombinant” are nucleic acids which have been produced byrecombinant DNA methodology, including those nucleic acids that aregenerated by procedures which rely upon a method of artificialrecombination, such as the polymerase chain reaction (PCR) and/orcloning into a vector using restriction enzymes. “Recombinant” nucleicacids are also those that result from recombination events that occurthrough the natural mechanisms of cells, but are selected for after theintroduction to the cells of nucleic acids designed to allow and makeprobable a desired recombination event.

In one embodiment, the nucleic acid or portion thereof encodes a proteinor polypeptide having at least one function characteristic of amammalian CXCR3 protein (e.g., a human CXCR3 receptor), such as abinding activity (e.g., ligand, inhibitor and/or promoter binding), asignalling activity (e.g., activation of a mammalian G protein,induction of rapid and transient increase in the concentration ofcytosolic free calcium [Ca²⁺]_(i)), and/or stimulation of a cellularresponse (e.g., stimulation of chemotaxis, exocytosis or inflammatorymediator release by leukocytes). The present invention also relates morespecifically to isolated and/or recombinant nucleic acids or a portionthereof comprising sequences which encode a mammalian CXCR3 receptor ora portion thereof. The present invention relates even more specificallyto isolated and/or recombinant nucleic acids comprising sequences whichencode a human CXCR3 protein.

The invention further relates to isolated and/or recombinant nucleicacids, including double or single stranded DNA or RNA, that arecharacterized by (1) their ability to hybridize to: (a) a nucleic acidhaving the sequence SEQ ID NO:1, (b) a nucleic acid having a sequencewhich is complementary to SEQ ID NO:1, or (c) a portion of the foregoingcomprising the open reading frame of SEQ ID NO:1 (a portion of thestrand illustrated in FIG. 1 or the corresponding portion of thecomplementary strand); and/or (2) by their ability to encode apolypeptide having the amino acid sequence SEQ ID NO:2 or a functionalequivalent thereof (i.e., a polypeptide having ligand binding activityfor one or more natural or physiological ligand(s) of the receptorand/or stimulatory function responsive to ligand binding, such that itcan induce a cellular response (e.g., induction (including triggering orstimulation) of chemotaxis, exocytosis or inflammatory mediator releaseby leukocytes); and/or (3) by both characteristics.

In one embodiment, the percent amino acid sequence identity between SEQID NO:2 and functional equivalents thereof is at least about 60% (≧60%).In a preferred embodiment, functional equivalents of SEQ ID NO:2 shareat least about 70% sequence identity with SEQ ID NOS:2. More preferably,the percent amino acid sequence identity between SEQ ID NO:2 andfunctional equivalents thereof is at least about 80%, and still morepreferably, at least about 90%.

Isolated and/or recombinant nucleic acids meeting these criteriacomprise nucleic acids having sequences identical to sequences ofnaturally occurring mammalian CXCR3 receptors and portions thereof, orvariants of the naturally occurring sequences. Such variants includemutants differing by the addition, deletion or substitution of one ormore residues, modified nucleic acids in which one or more residues ismodified (e.g., DNA or RNA analogs), and mutants comprising one or moremodified residues. In one embodiment, the nucleic acid shares at leastabout 50% nucleotide sequence similarity, more preferably at least about75% nucleotide sequence similarity, and still more preferably at leastabout 90% nucleotide sequence similarity, with one strand of thesequence illustrated in SEQ ID NO:1 or to the coding region thereof.Preferred nucleic acids have lengths of at least about 40 nucleotides,more preferably at least about 50, and still more preferably at leastabout 75 nucleotides.

Such nucleic acids can be detected and isolated by hybridization underhigh stringency conditions or moderate stringency conditions, forexample. “High stringency conditions” and “moderate stringencyconditions” for nucleic acid hybridizations are explained on pages2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in CurrentProtocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 1,Suppl. 26, 1991), the teachings of which are incorporated herein byreference. Factors such as probe length, base composition, percentmismatch between the hybridizing sequences, temperature and ionicstrength influence the stability of nucleic acid hybrids. Thus, high ormoderate stringency conditions can be determined empirically.

Isolated and/or recombinant nucleic acids that are characterized bytheir ability to hybridize to a nucleic acid having the sequence SEQ IDNO:1 or the complement thereof (e.g., under high or moderate stringencyconditions) may further encode a protein or polypeptide having at leastone function characteristic of a mammalian CXCR3 protein (e.g., a humanCXCR3 protein), such as a binding activity (e.g., ligand, inhibitorand/or promoter binding), a signalling activity (e.g., activation of amammalian G protein, induction of rapid and transient increase in theconcentration of cytosolic free calcium [Ca²⁺]_(i)), and/or stimulationof a cellular response (e.g., stimulation of chemotaxis, exocytosis orinflammatory mediator release by leukocytes).

The human CXCR3 nucleic acid described herein, or sufficient portionsthereof, whether isolated, recombinant and/or synthetic, includingfragments produced by PCR, can be used as probes or primers to detectand/or recover nucleic acids (e.g., genomic DNA, allelic variants, cDNA)encoding CXCR3 receptors (homologs) or other related receptor genes(e.g., novel CXC chemokine receptor genes) from other mammalian speciesincluding, but not limited to primates (e.g., a primate other than ahuman, such as a monkey (e.g., cynomolgus monkey)), bovine, ovine,equine, canine, feline and rodent (e.g., guinea pig, murine species suchas rat, mouse). This can be achieved using the procedures describedherein or other suitable methods, including hybridization, PCR or othersuitable techniques. Mammalian nucleic acids can be used to prepareconstructs (e.g., vectors), receptor or fragments thereof, and hoststrains useful in the production and methods of use of receptor.

In one embodiment, a nucleic acid encoding a mammalian CXCR3 protein (orvariant) is producible by methods such as PCR amplification. Forexample, appropriate primers (e.g., a pair of primers or nested primers)can be designed which comprise a sequence which is complementary orsubstantially complementary to a portion of the human CXCR3 cDNAdescribed herein. For instance, primers-complementary to the 5′- or3′-ends of the coding sequence and/or flanking the coding sequence canbe designed. Such primers can be used in a polymerase chain reactionwith a suitable template nucleic acid to obtain nucleic acid encoding amammalian CXCR3, for example. Suitable templates include e.g.,constructs described herein (such as pcDNA3-Clone8), a cDNA or genomiclibrary or another suitable source of mammalian (e.g., a human, primate)cDNA or genomic DNA. Primers can contain portions complementary toflanking sequences of a construct selected as template as appropriate.

The binding function of a protein or polypeptide (e.g., encoded byhybridizing nucleic acid) can be detected in binding or bindinginhibition assays, using membrane fractions containing receptor or cellsexpressing receptor, for example (see e.g., Van Riper et al., J. Exp.Med., 177: 851-856 (1993); Sledziewski et al., U.S. Pat. No. 5,284,746(Feb. 8, 1994)). Thus, the ability of the encoded protein or polypeptideto bind a ligand, such as IP-10 or Mig, an inhibitor and/or promoter,can be assessed. The antigenic properties of proteins or polypeptidesencoded by nucleic acids of the present invention can be determined byimmunological methods employing antibodies that bind to a mammalianCXCR3, such as immunoblotting, immunoprecipitation and immunoassay(e.g., radioimmunoassay, ELISA).

The signalling function of a protein or polypeptide (e.g., encoded byhybridizing nucleic acid) can be detected by enzymatic assays for Gprotein activity responsive to receptor binding (e.g., exchange of GTPfor GDP on the G protein α subunit, using membrane fractions). G proteincoupling can be further assessed, for example, using assays in whichstimulation by G protein is blocked by treatment or pre-treatment ofcells or a suitable cellular fraction (e.g., membranes) with specificinhibitors of G proteins, such as Bordetella pertussis toxin (Bischoff,S. C. et al., Eur. J. Immunol., 23: 761-767 (1993); Sozzani, S. et al.,J. Immunol., 147: 2215-2221 (1991)).

The stimulatory function of a protein or polypeptide (e.g., encoded byhybridizing nucleic acid) can be detected by standard assays forchemotaxis or mediator release, using cells expressing the protein orpolypeptide (e.g., assays which monitor chemotaxis, exocytosis (e.g.,degranulation of enzymes, such as esterases (e.g., serine esterases),perforin, granzymes) or mediator release (e.g., histamine, leukotriene)in response to a ligand (e.g., a chemokine such as IP-10 or Mig) or apromoter (see e.g., Taub, D. D. et al., J. Immunol., 155: 3877-3888(1995); Baggliolini, M. and C. A. Dahinden, Immunology Today, 15:127-133 (1994) and references cited therein). Functions characteristicof a mammalian CXCR3 receptor may also be assessed by other suitablemethods.

These methods, alone or in combination with other suitable methods canalso be used in procedures for the identification and/or isolation ofnucleic acids which encode a polypeptide having the amino acid sequenceSEQ ID NO:2 or functional equivalents thereof, and having an activitydetected by the assay. Portions of isolated nucleic acids which encodepolypeptide portions of SEQ ID NO:2 having a certain function can bealso identified and isolated in this manner.

Nucleic acids of the present invention can be used in the production ofproteins or polypeptides. For example, a nucleic acid containing all orpart of the coding sequence for a mammalian CXCR3 receptor, or DNA whichhybridizes to the sequence SEQ ID NO:1, or the complement thereof, canbe incorporated into a construct for further manipulation of sequencesor for production of the encoded polypeptide in suitable host cells.Nucleic acids of the present invention can also be modified, forexample, by incorporation of or attachment (directly or indirectly) of adetectable label such as a radioisotope, spin label, antigen or enzymelabel, flourescent or chemiluminesent group and the like, and suchmodified forms are included within the scope of the invention.

Antisense Constructs

In another embodiment, the nucleic acid is an antisense nucleic acid,which is complementary, in whole or in part, to a target moleculecomprising a sense strand, and can hybridize with the target molecule.The target can be DNA, or its RNA counterpart (i.e., wherein T residuesof the DNA are U residues in the RNA counterpart). When introduced intoa cell using suitable methods, antisense nucleic acid can inhibit theexpression of the gene encoded by the sense strand. Antisense nucleicacids can be produced by standard techniques.

In one embodiment, the antisense nucleic acid is wholly or partiallycomplementary to and can hybridize with a target nucleic acid, whereinthe target nucleic acid can hybridize to a nucleic acid having thesequence of the complement of SEQ ID NO:1. For example, antisensenucleic acid can be complementary to a target nucleic acid having thesequence of SEQ ID NO:1 or a portion thereof sufficient to allowhybridization. In another embodiment, the antisense nucleic acid iswholly or partially complementary to and can hybridize with a targetnucleic acid which encodes a mammalian CXCR3 receptor (e.g., humanIP-10/Mig receptor CXCR3).

Antisense nucleic acids are useful for a variety of purposes, includingresearch and therapeutic applications. For example, a constructcomprising an antisense nucleic acid can be introduced into a suitablecell to inhibit receptor expression. Such a cell provides a valuablecontrol cell, for instance in assessing the specificity ofreceptor-ligand interaction with the parent cell or other related celltypes. In another aspect, such a construct can be introduced into someor all of the cells of a mammal. The antisense nucleic acid inhibitsreceptor expression, and inflammatory processes mediated by CXCR3receptors in the cells containing the construct can be inhibited. Thus,an inflammatory disease or condition can be treated using an antisensenucleic acid of the present invention. Suitable laboratory animalscomprising an antisense construct can also provide useful models fordeficiencies of leukocyte function, and of activated T lymphocytedeficiency in particular, and can provide further information regardingCXCR3 receptor function. Such animals can provide valuable models ofinfectious disease or cancer, useful for elucidating the role ofleukocytes, such as T lymphocytes and NK cells, in host defenses.

Method of Producing Recombinant Proteins

Another aspect of the invention relates to a method of producing amammalian CXCR3 protein or variant (e.g., portion) thereof. Recombinantprotein can be obtained, for example, by the expression of a recombinantnucleic acid (e.g., DNA) molecule encoding a mammalian CXCR3 or variantthereof in a suitable host cell, for example.

Constructs (e.g., expression vectors) suitable for the expression of amammalian CXCR3 protein or variant thereof are also provided. Theconstructs can be introduced into a suitable host cell, and cells whichexpress a recombinant mammalian CXCR3 protein or variant thereof can beproduced and maintained in culture. Such cells are useful for a varietyof purposes, including use in the production of protein forcharacterization, isolation and/or purification, (e.g., affinitypurification), use as immunogen, and in binding assays or otherfunctional assays (e.g., to screen for ligands, inhibitors and/orpromoters of receptor function), for instance. Suitable host cells canbe procaryotic, including bacterial cells such as E. coli, B. subtilisand or other suitable bacteria, or eucaryotic, such as fungal or yeastcells (e.g., Pichia pastoris, Aspergillus species, Saccharomycescerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or otherlower eucaryotic cells, and cells of higher eucaryotes such as thosefrom insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor, publishedNov. 24, 1994)) or mammals (e.g., Chinese hamster ovary cells (CHO), COScells, HuT 78 cells, 293 cells). (See, e.g., Ausubel, F. M. et al., eds.Current Protocols in Molecular Biology, Greene Publishing Associates andJohn Wiley & Sons Inc., (1993)).

Host cells which produce a recombinant mammalian CXCR3 protein orvariant thereof can be produced as follows. For example, a nucleic acidencoding all or part of the coding sequence for the desired protein canbe inserted into a nucleic acid vector, e.g., a DNA vector, such as aplasmid, virus or other suitable replicon for expression. A variety ofvectors are available, including vectors which are maintained in singlecopy or multiple copy, or which become integrated into the host cellchromosome.

Transcriptional and/or translational signals of a mammalian CXCR3 genecan be used to direct expression. Suitable expression vectors for theexpression of a nucleic acid encoding all or part of the coding sequenceof the desired protein are also available. Suitable expression vectorscan contain a number of components, including, but not limited to one ormore of the following: an origin of replication; a selectable markergene; one or more expression control elements, such as a transcriptionalcontrol element (e.g., a promoter, an enhancer, terminator), and/or oneor more translation signals; a signal sequence or leader sequence formembrane targeting in a selected host cell (e.g., of mammalian origin orfrom a heterologous mammalian or non-mammalian species). In a construct,a signal sequence can be provided by the vector, the mammalian CXCR3coding sequence, or other source.

A promoter can be provided for expression in a suitable host cell.Promoters can be constitutive or inducible. In the vectors, a promotercan be operably linked to a nucleic acid encoding the mammalian CXCR3protein or variant thereof, such that it directs expression of theencoded polypeptide. A variety of suitable promoters for procaryotic(e.g., lac, tac, T3, T7 promoters for E. coli) and eucaryotic (e.g.,yeast alcohol dehydrogenase (ADH1), SV40, CMV) hosts are available.

In addition, the expression vectors typically comprise a selectablemarker for selection of host cells carrying the vector, and, in the caseof replicable expression vector, an origin or replication. Genesencoding products which confer antibiotic or drug resistance are commonselectable markers and may be used in procaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated. The present invention alsorelates to cells carrying these expression vectors.

For example, a nucleic acid encoding a mammalian CXCR3 protein orvariant thereof, or a construct comprising such nucleic acid, can beintroduced into a suitable host cell by a method appropriate to the hostcell selected (e.g., transformation, transfection, electroporation,infection), such that the nucleic acid is operably linked to one or moreexpression control elements (e.g., in a vector, in a construct createdby processes in the cell, integrated into the host cell genome). Hostcells can be maintained under conditions suitable for expression (e.g.,in the presence of inducer, suitable media supplemented with appropriatesalts, growth factors, antibiotic, nutritional supplements, etc.),whereby the encoded polypeptide is produced. If desired, the encodedprotein (e.g., human CXCR3) can be isolated (e.g., from the host cells,medium, milk). It will be appreciated that the method encompassesexpression in a host cell of a transgenic animal (see e.g., WO 92/03918,GenPharm International, published Mar. 19, 1992).

Fusion proteins can also be produced in this manner. For example, someembodiments can be produced by the insertion of a mammalian CXCR3protein cDNA or portion thereof into a suitable expression vector, suchas Bluescript®II SK +/− (Stratagene), pGEX-4T-2 (Pharmacia), pcDNA-3(Invitrogen) or pET-15b (Novagen). The resulting construct can beintroduced into a suitable host cell for expression. Upon expression,fusion protein can be isolated or purified from a cell lysate by meansof a suitable affinity matrix (see e.g., Current Protocols in MolecularBiology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26, pp.16.4.1-16.7.8 (1991)). In addition, affinity labels provide a means ofdetecting a fusion protein. For example, the cell surface expression orpresence in a particular cell fraction of a fusion protein comprising anantigen or epitope affinity label can be detected by means of anappropriate antibody.

Antibodies

The invention further relates to antibodies reactive with a mammalianCXCR3 protein or portion thereof. In one embodiment, antibodies areraised against an isolated and/or recombinant mammalian CXCR3 protein orportion thereof (e.g., a peptide) or against a host cell which expressesrecombinant mammalian CXCR3. In a preferred embodiment, antibodiesspecifically bind mammalian CXCR3 receptor(s) or a portion thereof, andin a particularly preferred embodiment the antibodies can inhibit(reduce or prevent) the interaction of receptor with a natural ligand,such as IP-10 and/or Mig.

The antibodies of the present invention can be polyclonal or monoclonal,and the term antibody is intended to encompass both polyclonal andmonoclonal antibodies. The terms polyclonal and monoclonal refer to thedegree of homogeneity of an antibody preparation, and are not intendedto be limited to particular methods of production.

Antibodies of the present invention can be raised against an appropriateimmunogen, including proteins or polypeptides of the present invention,such as isolated and/or recombinant mammalian CXCR3 protein or portionthereof (including synthetic molecules, such as synthetic peptides). Inaddition, cells expressing recombinant mammalian CXCR3, such astransfected cells, can be used as immunogens or in a screen for antibodywhich binds receptor. See for example, Chuntharapai et al., J. Immunol.,152: 1783-1789 (1994); Chuntharapai et al., U.S. Pat. No. 5,440,021).

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11 (1991)). Generally, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0)with antibody producing cells. The antibody producing cell, preferablythose of the spleen or lymph nodes, can be obtained from animalsimmunized with the antigen of interest. The fused cells (hybridomas) canbe isolated using selective culture conditions, and cloned by limitingdilution. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can used, including, for example, methods whichselect recombinant antibody from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies (see e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani etal., U.S. Pat. No. 5,545,807).

Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted), or veneered antibodies, as well as chimeric, CDR-graftedor veneerd single chain antibodies, comprising portions derived fromdifferent species, and the like are also encompassed by the presentinvention and the term “antibody”. The various portions of theseantibodies can be joined together chemically by conventional techniques,or can be prepared as a contiguous protein using genetic engineeringtechniques. For example, nucleic acids encoding a chimeric or humanizedchain can be expressed to produce a contiguous protein. See, e.g.,Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen etal., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0519 596 A1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460(1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988))regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments ofchimeric, humanized, primatized, veneered or single chain antibodies,can also be produced. Functional fragments of foregoing antibodiesretain at least one binding function and/or modulation function of thefull-length antibody from which they are derived. For example, antibodyfragments capable of binding to a mammalian CXCR3 protein or portionthereof, including, but not limited to, Fv, Fab, Fab′ and F(ab′)₂fragments are encompassed by the invention. Such fragments can beproduced by enzymatic cleavage or by recombinant techniques. Forinstance, papain or pepsin cleavage can generate Fab or F(ab′)₂fragments, respectively. Antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain portion can be designed toinclude DNA sequences encoding the CH₁ domain and hinge region of theheavy chain.

The antibodies of the present invention are useful in a variety ofapplications, including research, diagnostic and therapeuticapplications. For instance, they can be used to isolate and/or purifyreceptor or portions thereof, and to study receptor structure (e.g.,conformation) and function.

The antibodies of the present invention can also be used to modulatereceptor function in research and therapeutic applications. Forinstance, antibodies can act as inhibitors to inhibit (reduce orprevent) (a) binding (e.g., of a ligand, a second inhibitor or apromoter) to the receptor, (b) receptor signalling, and/or (c) acellular response. Antibodies which act as inhibitors of receptorfunction can block ligand or promoter binding directly or indirectly(e.g., by causing a conformational change in the receptor). For example,antibodies can inhibit receptor function by inhibiting binding of aligand, or by desensitization (with or without inhibition of binding ofa ligand).

Antibodies which bind receptor can also act as agonists of receptorfunction, triggering or stimulating a receptor function, such assignalling and/or a cellular response (e.g., calcium flux, chemotaxis,exocytosis or pro-inflammatory mediator release) upon binding toreceptor.

In addition, the various antibodies of the present invention can be usedto detect or measure the expression of receptor, for example, onleukocytes such as activated T cells or natural killer cells (NK cells),or on cells transfected with a receptor gene. Thus, they also haveutility in applications such as cell sorting (e.g., flow cytometry,fluorescence activated cell sorting), for diagnostic or researchpurposes.

Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodiesrecognize antigenic determinants associated with the antigen-bindingsite of another antibody. Anti-idiotypic antibodies can be prepared aagainst a first antibody by immunizing an animal of the same species,and preferably of the same strain as the animal used to produce thefirst antibody, with said first antibody. See e.g., U.S. Pat. No.4,699,880.

In one embodiment, antibodies are raised against receptor or a portionthereof, and these antibodies are used in turn as immunogen to producean anti-idiotypic antibody. The anti-Id produced thereby can mimicreceptor and bind compounds which bind receptor, such as ligands,inhibitors or promoters of receptor function, and can be used in animmunoassay to detect, identify or quantitate such compounds. Such ananti-idiotypic antibody can also be an inhibitor of receptor function,although it does not bind receptor itself.

Anti-idiotypic (i.e., Anti-Id) antibody can itself be used to raise ananti-idiotypic antibody (i.e., Anti-anti-Id). Such an antibody can besimilar or identical in specificity to the original immunizing antibody.In one embodiment, antibody antagonists which block binding to receptorcan be used to raise Anti-Id, and the Anti-Id can be used to raiseAnti-anti-Id, which can have a specificity which is similar or identicalto that of the antibody antagonist. These anti-anti-Id antibodies can beassessed for inhibitory effect on receptor function to determine if theyare antagonists.

Single chain, and chimeric, humanized, primatized (CDR-grafted),veneered, as well as chimeric, CDR-grafted, or veneered single chainanti-idiotypic antibodies can be prepared, and are encompassed by theterm anti-idiotypic antibody. Antibody fragments of such antibodies canalso be prepared.

The antibodies and fragments of the present invention can be modified,for example, by incorporation of or attachment (directly or indirectly)of a detectable label such as a radioisotope, spin label, antigen orenzyme label, flourescent or chemiluminesent group and the like, andsuch modified forms are included within the scope of the invention.

Identification of Ligands, Inhibitors or Promoters of Receptor Function

As used herein, a ligand is a substance which binds to a receptorprotein. A ligand of a selected mammalian CXCR3 protein is a substancewhich binds to the selected mammalian CXCR3 protein. In a preferredembodiment, ligand binding of a mammalian CXCR3 protein occurs with highaffinity. The term ligand refers to substances including, but notlimited to, a natural ligand, whether isolated and/or purified,synthetic, and/or recombinant, a homolog of a natural ligand (e.g., fromanother mammal), antibodies, portions of such molecules, and othersubstances which bind receptor. A natural ligand of a selected mammalianreceptor can bind to the receptor under physiological conditions, and isof a mammalian origin which is the same as that of the mammalian CXCR3protein. The term ligand encompasses substances which are inhibitors orpromoters of receptor activity, as well as substances which bindreceptor, but lack inhibitor or promoter activity.

As used herein, an inhibitor is a substance which inhibits at least onefunction characteristic of a mammalian CXCR3 protein (e.g., a humanCXCR3), such as a binding activity (e.g., ligand binding, promoterbinding), a signalling activity (e.g., activation of a mammalian Gprotein, induction of rapid and transient increase in the concentrationof cytosolic free calcium [Ca²⁺]_(i)), and/or cellular response function(e.g., stimulation of chemotaxis, exocytosis or inflammatory mediatorrelease by leukocytes). The term inhibitor refers to substancesincluding antagonists which bind receptor (e.g., an antibody, a mutantof a natural ligand, other competitive inhibitors of ligand binding),and substances which inhibit receptor function without binding thereto(e.g., an anti-idiotypic antibody).

As used herein, a promoter is a substance which promotes (induces orenhances) at least one function characteristic of a mammalian CXCR3protein (e.g., a human CXCR3), such as a binding activity (e.g., ligand,inhibitor and/or promoter binding), a signalling activity (e.g.,activation of a mammalian G protein, induction of rapid and transientincrease in the concentration of cytosolic free calcium [Ca²⁺]_(i)),and/or a cellular response function (e.g., stimulation of chemotaxis,exocytosis or inflammatory mediator release by leukocytes). The termpromoter refers to substances including agonists which bind receptor(e.g., an antibody, a homolog of a natural ligand from another species),and substances which promote receptor function without binding thereto(e.g., by activating an associated protein). In a preferred embodiment,the agonist is other than a homolog of a natural ligand.

The assays described below, which rely upon the nucleic acids andproteins of the present invention, can be used, alone or in combinationwith each other or other suitable methods, to identify ligands,inhibitors or promoters of a mammalian CXCR3 protein or variant. The invitro methods of the present invention can be adapted forhigh-throughput screening in which large numbers of samples areprocessed (e.g., a 96 well format). Host cells comprising a nucleic acidof the present invention and expressing recombinant mammalian CXCR3(e.g., human CXCR3) at levels suitable for high-throughput screening canbe used, and thus, are particularly valuable in the identificationand/or isolation of ligands, inhibitors and promoters of mammalian CXCR3proteins. Expression of receptor can be monitored in a variety of ways.For instance, expression can be monitored using antibodies of thepresent invention which bind receptor or a portion thereof. Also,commercially available antibodies can be used to detect expression of anantigen- or epitope-tagged fusion protein comprising a receptor proteinor polypeptide (e.g., FLAG tagged receptors), and cells expressing thedesired level can be selected.

Nucleic acid encoding a mammalian CXCR3 protein, the can be incorporatedinto an expression system to produce a receptor protein or polypeptideas described above. An isolated and/or recombinant receptor protein orpolypeptide, such as a receptor expressed in cells stably or transientlytransfected with a construct comprising a nucleic acid of the presentinvention, or in a cell fraction containing receptor (e.g., a membranefraction from transfected cells, liposomes incorporating receptor), canbe used in tests for receptor function. The receptor can be furtherpurified if desired. Testing of receptor function can be carried out invitro or in vivo.

An isolated, recombinant mammalian CXCR3 protein, such as a human CXCR3as shown in FIG. 2 (SEQ ID NO:2), can be used in the present method, inwhich the effect of a compound is assessed by monitoring receptorfunction as described herein or using other suitable techniques. Forexample, stable or transient transfectants such as those described inExample 2 or other suitable cells (e.g., baculovirus infected Sf9 cells,stable tranfectants of mouse L1-2 pre-B cells (derived from a pre-Blymphoma, Dr. Eugene Butcher (Stanford University, Stanford, Calif.)),can be used in binding assays. Stable transfectants of Jurkat cells(Example 2) or of other suitable cells capable of chemotaxis can be used(e.g., mouse L1-2 pre-B cells) in chemotaxis assays, for example.

According to the method of the present invention, compounds can beindividually screened or one or more compounds can be testedsimultaneously according to the methods herein. Where a mixture ofcompounds is tested, the compounds selected by the processes describedcan be separated (as appropriate) and identified by suitable methods(e.g., PCR, sequencing, chromatography). The presence of one or morecompounds (e.g., a ligand, inhibitor, promoter) in a test sample canalso be determined according to these methods.

Large combinatorial libraries of compounds (e.g., organic compounds,recombinant or synthetic peptides, “peptoids”, nucleic acids) producedby combinatorial chemical synthesis or other methods can be tested (seee.g., Zuckerman, R. N. et al., J. Med. Chem., 37: 2678-2685 (1994) andreferences cited therein; see also, Ohlmeyer, M. H. J. et al., Proc.Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al.,Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to taggedcompounds; Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D.et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092). Where compounds selected from a combinatorial library by thepresent method carry unique tags, identification of individual compoundsby chromatographic methods is possible.

In one embodiment, phage display methodology is used. For example,receptor can be contacted with a phage (e.g., a phage or collection ofphage such as a library) displaying a polypeptide under conditionsappropriate for receptor binding (e.g., in a suitable binding buffer).Phage bound to receptor can be selected using standard techniques orother suitable methods. Phage can be separated from receptor using asuitable elution buffer. For example, a change in the ionic strength orpH can lead to a release of phage. Alternatively, the elution buffer cancomprise a release component or components designed to disrupt bindingof compounds (e.g., one or more compounds which can disrupt binding ofthe displayed peptide to the receptor, such as a ligand, inhibitor,and/or promoter which competitively inhibits binding). Optionally, theselection process can be repeated or another selection step can be usedto further enrich for phage which bind receptor. The displayedpolypeptide can be characterized (e.g., by sequencing phage DNA). Thepolypeptides identified can be produced and further tested for ligandbinding, inhibitor and/or promoter function. Analogs of such peptidescan be produced which will have increased stability or other desirableproperties.

In one embodiment, phage expressing and displaying fusion proteinscomprising a coat protein with an N-terminal peptide encoded by randomsequence nucleic acids can be produced. Suitable host cells expressing areceptor protein or polypeptide of the present invention are contactedwith the phage, bound phage are selected, recovered and characterized.(See e.g., Doorbar, J. and G. Winter, J. Mol. Biol., 244: 361 (1994)discussing a phage display procedure used with a G protein-coupledreceptor).

Other sources of potential ligands, inhibitors and/or promoters ofmammalian CXCR3 proteins include, but are not limited to, variants ofCXCR3 ligands, including naturally occurring, synthetic or recombinantvariants of IP-10 or Mig, substances such as other chemoattractants orchemokines, variants thereof, other inhibitors and/or promoters (e.g.,anti-CXCR3 antibodies, antagonists, agonists), other G-protein coupledreceptor ligands, inhibitors and/or promoters (e.g., antagonists oragonists), and soluble portions of a mammalian CXCR3 receptor, such as asuitable receptor peptide or analog which can inhibit receptor function(see e.g., Murphy, R. B., WO 94/05695).

Binding Assays

The isolated and/or recombinant receptor proteins, portions thereof, orsuitable fusion proteins of the present invention, can be used in amethod to select and identify agents which bind to a (one or more)mammalian CXCR3 protein, such as human CXCR3, and which are ligands, orpotential inhibitors or promoters of receptor activity. Agents selectedby the method, including ligands, inhibitors or promoters, can befurther assessed for an inhibitory or stimulatory effect on receptorfunction and/or for therapeutic utility.

In one embodiment, an agent which binds to an active, isolated and/orrecombinant mammalian CXCR3 protein or polypeptide is identified by themethod. In this embodiment, the receptor protein or polypeptide used hasat least one property, activity or function characteristic of amammalian CXCR3 protein (as defined herein), such as a binding activity(e.g., ligand, inhibitor and/or promoter binding), a signalling activity(e.g., activation of a mammalian G protein, induction of rapid andtransient increase in the concentration of cytosolic free calcium[Ca²⁺]_(i)), cellular response function (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes),and/or an immunological property as defined herein. In a preferredembodiment, the isolated and/or recombinant mammalian CXCR3 protein orvariant has ligand binding function, and more preferably binds a naturalligand of the receptor. In a particularly preferred embodiment, theisolated and/or recombinant protein is a human CXCR3 protein encoded bythe nucleic acid illustrated FIG. 1 (SEQ ID NO:1).

For example, a composition comprising an isolated and/or recombinantmammalian CXCR3 protein or variant thereof can be maintained underconditions suitable for binding, the receptor can be contacted with anagent (e.g., a composition comprising one or more agent) to be tested,and binding is detected or measured. In one embodiment, a receptorprotein can be expressed in cells stably or transiently transfected witha construct comprising a nucleic acid sequence which encodes a receptorof the present invention. The cells can be maintained under conditionsappropriate for expression of receptor. The cells are contacted with anagent under conditions suitable for binding (e.g., in a suitable bindingbuffer), and binding can be detected by standard techniques. Forexample, the extent of binding can be determined relative to a suitablecontrol (e.g., compared with background determined in the absence ofagent, compared with binding of a second agent (i.e., a standard),compared with binding of the agent to untransfected cells). Optionally,a cellular fraction, such as a membrane fraction, containing receptorcan be used in lieu of whole cells.

Binding or complex formation can be detected directly or indirectly. Inone embodiment, the agent can be labeled with a suitable label (e.g.,fluorescent label, chemiluminescent label, isotope label, enzyme label),and binding can be determined by detection of the label. Specificity ofbinding can be assessed by competition or displacement, for example,using unlabeled agent or a ligand (e.g., IP-10, Mig) as competitor.

Ligands of the mammalian receptor, including natural ligands from thesame mammalian species or from another species, can be identified inthis manner. The binding activity of a promoter or inhibitor which bindsreceptor can also be assessed using such a ligand binding assay.

Binding inhibition assays can also be used to identify ligands, andinhibitors and promoters which bind receptor and inhibit binding ofanother agent such as a ligand. For example, a binding assay can beconducted in which a reduction in the binding of a first agent (in theabsence of a second agent), as compared with binding of the first agentin the presence of the second test agent, is detected or measured. Thereceptor can be contacted with the first and second agentssimultaneously, or one after the other, in either order. A reduction inthe extent of binding of the first agent in the presence of the secondtest agent, is indicative of inhibition of binding by the second agent.For example, binding of the first agent could be decreased or abolished.

In one embodiment, direct inhibition of the binding of a first agent(e.g., a chemokine such as IP-10, Mig) to a human CXCR3 by a second testagent is monitored. For example, the ability of an agent to inhibit thebinding of ¹²⁵I-labeled Mig to human CXCR3 can be monitored. Such anassay can be conducted using whole cells (e.g., a suitable cell linecontaining nucleic acid encoding a human CXCR3 receptor), or a membranefraction from said cells, for instance.

Other methods of identifying the presence of an agent(s) which binds areceptor are available, such as methods which monitor events which aretriggered by receptor binding, including signalling function and/orstimulation of a cellular response.

It will be understood that the inhibitory effect of antibodies of thepresent invention can be assessed in a binding inhibition assay.Competition between antibodies for receptor binding can also be assessedin the method in which the first agent in the assay is another antibody,under conditions suitable for antibody binding.

Ligands, receptor-binding inhibitors and promoters, which are identifiedin this manner, can be further assessed to determine whether, subsequentto binding, they act to inhibit or activate other functions of CXCR3receptors and/or to assess their therapeutic utility.

Signalling Assays

The binding of a G protein-coupled receptor (e.g., by an agonist) canresult in signalling by the receptor, and stimulation of the activity ofG protein. The induction of signalling function by an agent can bemonitored using any suitable method. For example, G protein activity,such as hydrolysis of GTP to GDP, or later signalling events triggeredby receptor binding, such as induction of rapid and transient increasein the concentration of intracellular (cytosolic) free calcium[Ca²⁺]_(i), can be assayed by methods known in the art or other suitablemethods (Example 2; see also, Neote, K. et al., Cell, 72: 415-425 1993);Van Riper et al., J. Exp. Med., 177: 851-856 (1993); Dahinden, C. A. etal., J. Exp. Med., 179: 751-756 (1994)).

The functional assay of Sledziewski et al. using hybrid G proteincoupled receptors can also be used to identify a ligand or promoter byits ability to activate a hybrid G protein or to identify an inhibitorby its ability to inhibit such activation (Sledziewski et al., U.S. Pat.No. 5,284,746, the teachings of which are incorporated herein byreference). In one embodiment, a biological response of the host cell(triggered by binding to hybrid receptor) can be monitored, detection ofthe response being indicative of the presence of ligand in the testsample. For example, a method of detecting the presence of a ligand in atest sample is described, wherein the ligand is an agent which iscapable of being bound by the ligand-binding domain of a receptor. Inone embodiment of the method, yeast host cells are transformed with aDNA construct capable of directing the expression of a biologicallyactive hybrid G protein-coupled receptor (i.e., a fusion protein). Thehybrid receptor comprises a mammalian G protein-coupled receptor havingat least one domain other than the ligand-binding domain replaced with acorresponding domain of a yeast G protein-coupled receptor, such as aSTE2 gene product. The yeast host cells containing the construct aremaintained under conditions in which the hybrid receptor is expressed,and the cells are contacted with a test sample under conditions suitableto permit binding of ligand to the hybrid receptor. A biologicalresponse of the host cell (triggered by binding to hybrid receptor) ismonitored, detection of the response being indicative of a signallingfunction. For instance, binding to a hybrid receptor derived from STE2gene product can lead to induction of the BAR1 promoter. Induction ofthe promoter can be measured by means of a reporter gene (e.g., β-gal),which is linked to the BAR1 promoter and introduced into host cells on asecond construct. Expression of the reporter gene can be detected by anin vitro enzyme assay on cell lysates or by the presence of bluecolonies on plates containing an indicator (e.g., X-gal) in the medium,for example.

In another embodiment, the assay can be used to identify potentialinhibitors of receptor function. The inhibitory activity of an agent canbe determined using a ligand or promoter in the assay, and assessing theability of the test agent to inhibit the activity induced by ligand orpromoter.

Variants of known ligands can also be screened for reduced ability(decreased ability or no ability) to stimulate activity of a coupled Gprotein. In this embodiment, although the agent has ligand bindingactivity (as determined by another method), engagement of the receptordoes not trigger or only weakly triggers activity of a coupled Gprotein. Such agents are potential antagonists, and can be furtherassessed for inhibitory activity.

Chemotaxis and Other Assays of Cellular Responses

Chemotaxis assays can also be used to assess receptor function. Theseassays are based on the functional migration of cells in vitro or invivo induced by an agent, and can be used to assess the binding and/oreffect on chemotaxis of ligands, inhibitors, or promoters.

The use of an in vitro chemotaxis assay to assess the response of cellsto IP-10 and Mig is described in Example 2. Springer et al. describe atransendothelial lymphocyte chemotaxis assay (Springer et al., WO94/20142, published Sep. 15, 1994, the teachings of which areincorporated herein by reference; see also Berman et al., ImmunolInvest., 17: 625-677 (1988)). Migration across endothelium into collagengels has also been described (Kavanaugh et al., J. Immunol, 146:4149-4156 (1991)).

Generally, chemotaxis assays monitor the directional movement ormigration of a suitable cell capable of chemotaxis, such as a leukocyte(e.g., T lymphocytes, NK cells, monocytes), stable transfectants ofJurkat cells, mouse L1-2 pre-B cells or of other suitable host cells,for example, into or through a barrier (e.g., endothelium, a filter),toward increased levels of an agent, from a first surface of the barriertoward an opposite second surface. Membranes or filters provideconvenient barriers, such that the directional movement or migration ofa suitable cell into or through a filter, toward increased levels of anagent, from a first surface of the filter toward an opposite secondsurface of the filter, is monitored. In some assays, the membrane iscoated with a substance to facilitate adhesion, such as ICAM-1,fibronectin or collagen.

For example, one can detect or measure the migration of cells in asuitable container (a containing means), from a first chamber into orthrough a microporous membrane into a second chamber which contains anagent to be tested, and which is divided from the first chamber by themembrane. A suitable membrane, having a suitable pore size formonitoring specific migration in response to the agent, including, forexample, nitrocellulose, polycarbonate, is selected. For example, poresizes of about 3-8 microns, and preferably about 5-8 microns can beused. Pore size can be uniform on a filter or within a range of suitablepore sizes.

To assess migration, the distance of migration into the filter, thenumber of cells crossing the filter that remain adherent to the secondsurface of the filter, and/or the number of cells that accumulate in thesecond chamber can be determined using standard techniques (e.g., bymicroscopy). In one embodiment, the cells are labeled with a detectablelabel (e.g., radioisotope, fluorescent label, antigen or epitope label),and migration can be assessed by determining the presence of the labeladherent to the membrane and/or present in the second chamber using anappropriate method (e.g., by detecting radioactivity, fluorescence,immunoassay). The extent of migration induced by an agent can bedetermined relative to a suitable control (e.g., compared to backgroundmigration determined in the absence of the agent, to the extent ofmigration induced by a second agent (i.e., a standard), compared withmigration of untransfected cells induced by the agent).

Chambers can be formed from various solids, such as plastic, glass,polypropylene, polystyrene, etc. Membranes which are detachable from thechambers, such as a Biocoat (Collaborative Biomedical Products) orTranswell (Costar, Cambridge, Mass.) culture insert, facilitate countingadherent cells. In the container, the filter can be situated so as to bein contact with fluid containing cells in the first chamber, and thefluid in the second chamber. Other than the test agent or additionalligand, inhibitor, or promoter present for the purpose of the assay, thefluid on either side of the membrane is preferably the same orsubstantially similar. The fluid in the chambers can comprise proteinsolutions (e.g., bovine serum albumin, fetal calf serum, human serumalbumin) which may act to increase stability and inhibit nonspecificbinding of cells, and/or culture media.

In one embodiment, transendothelial migration is assessed. In additionto lower background (signal to noise ratio), transendothelial migrationmodels in vivo conditions in which leukocytes emigrate from bloodvessels toward chemoattractants present in the tissues at sites ofinflammation by crossing the endothelial cell layer lining the vesselwall. In this embodiment, transmigration through an endothelial celllayer assessed. To prepare the cell layer, endothelial cells can becultured on a microporous filter or membrane, optionally coated with asubstance such as collagen, fibronectin, or other extracellular matrixproteins, to facilitate the attachment of endothelial cells. Preferably,endothelial cells are cultured until a confluent monolayer is formed. Avariety of mammalian endothelial cells can are available for monolayerformation, including for example, vein, artery or microvascularendothelium, such as human umbilical vein endothelial cells (CloneticsCorp, San Diego, Calif.) or a suitable cell line, such as the ECV 304cell line used (European Collection of Animal Cell Cultures, PortonDown, Salisbury, U.K.). To assay chemotaxis in response to a particularmammalian receptor, endothelial cells of the same mammal are preferred;however endothelial cells from a heterologous mammalian species or genuscan also be used.

Generally, the assay is performed by detecting the directional migrationof cells into or through a membrane or filter, in a direction towardincreased levels of an agent, from a first surface of the filter towardan opposite second surface of the filter, wherein the filter contains anendothelial cell layer on a first surface. Directional migration occursfrom the area adjacent to the first surface, into or through themembrane, towards an agent situated on the opposite side of the filter.The concentration of agent present in the area adjacent to the secondsurface, is greater than that in the area adjacent to the first surface.

In one embodiment, a chemotaxis assay is used to test for ligand orpromoter activity of an agent, a composition comprising cells capable ofmigration and expressing a mammalian CXCR3 protein are placed in thefirst chamber, and a composition comprising the agent (one or moreagents) to be tested is placed in the second chamber, preferably in theabsence of other ligands or promoters capable of inducing chemotaxis ofthe cells in the first chamber (having chemoattractant function).However, one or more ligands or promoters having chemoattractantfunction may be present. The ability of an agent to induce chemotaxis ofthe cells expressing a mammalian CXCR3 receptor in this assay isindicative that the agent is a ligand or promoter of receptor function.

In one embodiment used to test for an inhibitor, a compositioncomprising cells capable of migration and expressing a mammalian CXCR3protein are placed in the first chamber. A composition comprising aligand or promoter (i.e., one or more ligands or promoters) capable ofinducing chemotaxis of the cells in the first chamber (havingchemoattractant function) is placed in the second chamber. Before(preferably shortly before) the cells are placed in the first chamber,or simultaneously with the cells, a composition comprising the agent tobe tested is placed, preferably, in the first chamber. The ability of anagent to inhibit ligand- or promoter-induced chemotaxis of the cellsexpressing a mammalian CXCR3 protein in this assay is indicative thatthe agent is an inhibitor of receptor function (e.g., an inhibitor ofcellular response function). A reduction in the extent of migrationinduced by the ligand or promoter in the presence of the test agent, isindicative of inhibitory activity. Separate binding studies (see above)can be performed to determine whether inhibition is a result of bindingof the test agent to receptor or occurs via a different mechanism.

In vivo assays which monitor leukocyte infiltration of a tissue, inresponse to injection of an agent in the tissue, are described below.These models measure the ability of cells to respond to a ligand orpromoter by emigration and chemotaxis to a site of inflammation.

The effects of a ligand, inhibitor or promoter on the cellular responsefunction of a CXCR3 receptor can be assessed by monitoring othercellular responses induced by active receptor, using suitable host cellscontaining receptor. Similarly, these assays can be used to determinethe function of a receptor. For instance, exocytosis (e.g.,degranulation of natural killer cells leading to release of one or moreenzymes or other granule components, such as esterases (e.g., serineesterases), perforin, and/or granzymes), inflammatory mediator release(such as release of bioactive lipids such as leukotrienes (e.g.,leukotriene C₄)), and respiratory burst, can be monitored by methodsknown in the art or other suitable methods. (See e.g., Taub, D. D. etal., J. Immunol., 155: 3877-3888 (1995) regarding assays for release ofgranule-derived serine esterases (the teachings of which areincorporated herein by reference) and Loetscher et al., J. Immunol.,156: 322-327 (1996) regarding assays for enzyme and granzyme release byNK cells and cytotoxic T lymphocytes (CTLs) (the teachings of which areincorporated herein by reference); Rot, A. et al., J. Exp. Med., 176:1489-1495 (1992) regarding respiratory burst; Bischoff. S. C. et al.,Eur. J. Immunol., 23: 761-767 (1993) and Baggliolini, M. and C. A.Dahinden, Immunology Today, 15: 127-133 (1994)).

In one embodiment, a ligand, inhibitor and/or promoter is identified bymonitoring the release of an enzyme upon degranulation or exocytosis bya cell capable of this function. Cells containing a nucleic acid of thepresent invention, which encodes an active receptor protein capable ofstimulating exocytosis or degranulation are maintained in a suitablemedium under suitable conditions, whereby receptor is expressed anddegranulation can be induced. The receptor is contacted with an agent tobe tested, and enzyme release is assessed. The release of an enzyme intothe medium can be detected or measured using a suitable assay, such asin an immunological assay, or biochemical assay for enzyme activity.

The medium can be assayed directly, by introducing components of theassay (e.g., substrate, co-factors, antibody) into the medium (e.g.,before, simultaneous with or after the cells and agent are combined).Alternatively, the assay can be performed on medium which has beenseparated from the cells or further processed (e.g., fractionated) priorto assay. For example, convenient assays for are available for enzymessuch serine esterases (see e.g., Taub, D. D. et al., J. Immunol., 155:3877-3888 (1995) regarding release of granule-derived serine esterases).

Stimulation of degranulation by an agent can be indicative that theagent is a ligand or promoter of a mammalian CXCR3 protein. In anotherembodiment, cells expressing receptor are combined with a ligand orpromoter, and an agent to be tested is added before, after orsimultaneous therewith, and degranulation is assessed. Inhibition ofligand- or promoter-induced degranulation is indicative that the agentis an inhibitor of mammalian CXCR3 protein function.

Cellular adherence can also monitored by methods known in the art orother suitable methods. Engagement of the chemokine receptors of alymphocyte can cause integrin activation, and induction of adherence toadhesion molecules expressed in vasculature or the perivascular space.In one embodiment, a ligand, inhibitor and/or promoter is identified bymonitoring cellular adherence by a cell capable of adhesion. Forexample, an agent to be tested can be combined with (a) cells expressingreceptor (preferably non-adherent cells which when transfected withreceptor aquire adhesive ability), (b) a composition comprising asuitable adhesion molecule (e.g., a substrate such as a culture wellcoated with an adhesion molecule, such as fibronectin), and (c) a ligandor promoter (e.g., agonist), and maintained under conditions suitablefor ligand- or promoter-induced adhesion. Labeling of cells with afluorescent dye provides a convenient means of detecting adherent cells.Nonadherent cells can be removed (e.g., by washing) and the number ofadherent cells determined. The effect of the agent in inhibiting orenhancing ligand- or promoter-induced adhesion can be indicative ofinhibitor or promoter activity, respectively. Agents active in the assayinclude inhibitors and promoters of binding, signalling, and/or cellularresponses. In another embodiment, an agent to be tested can be combinedwith cells expressing receptor and a composition comprising a suitableadhesion molecule under conditions suitable for ligand- orpromoter-induced adhesion, and adhesion is monitored. Increased adhesionrelative to a suitable control is indicative of the presence of a ligandand/or promoter.

Models of Inflammation

A variety of in vivo models of inflammation are available, which can beused to assess the effects of ligands, inhibitors, or promoters in vivoas therapeutic agents, including a sheep model for asthma (see e.g.,Weg, V. B. et al., J. Exp. Med., 177: 561 (1993), the teachings of whichare incorporated herein by reference), a rat delayed typehypersensitivity model (Rand, M. L. et al., Am. J. Pathol., 148: 855-864(1996), the teachings of which are incorporated herein by reference), orother suitable models.

In addition, leukocyte infiltration upon intradermal injection of acompound into a suitable animal, such as rabbit, rat, or guinea pig, canbe monitored (see e.g., Van Damme J. et al., J. Exp. Med., 176: 59-65(1992); Zachariae, C. O. C. et al., J. Exp. Med., 171: 2177-2182 (1990);Jose, P. J. et al., J. Exp. Med., 179: 881-887 (1994)). In oneembodiment, skin biopsies are assessed histologically for infiltrationof leukocytes (e.g., T lymphocytes, monocytes, natural killer cells). Inanother embodiment, labeled cells (e.g., cells expressing a mammalianCXCR3 protein which are labeled with ¹¹¹In, for example) capable ofchemotaxis and extravasation are administered to the animal.Infiltration of labelled cells in the vicinity of the site of injectionof a test sample (e.g., a compound to be tested in a suitable buffer orphysiological carrier) is indicative of the presence of a ligand orpromoter, such as an agonist, in the sample. These assays can also bemodified to identify inhibitors of chemotaxis and leukocyteextravasation. For example, an inhibitor can be administered, eitherbefore, simultaneously with or after ligand or agonist is administeredto the test animal. A decrease of the extent of infiltration in thepresence of inhibitor as compared with the extent of infiltration in theabsence of inhibitor is indicative of inhibition.

Diagnostic Applications

The present invention has a variety of diagnostic applications. Forexample, a mutation(s) in a gene encoding a mammalian CXCR3 protein cancause a defect in at least one function of the encoded receptor, therebyreducing or enhancing receptor function. For instance, a mutation whichproduces a variant of receptor or alters the level of expression, canreduce or enhance receptor function, reducing or enhancing processesmediated by receptor (e.g., inflammatory processes). The presence ofsuch a mutation can be determined using methods which detect or measurethe presence of receptor or receptor function in cells (e.g.,leukocytes, such as activated T lymphocytes) of an individual. or in areceptor preparation isolated from such cells. In these assays, reducedor enhanced levels of receptor and/or reduced or enhanced receptorfunction can be assessed.

The nucleic acids of the present invention provide reagents, such asprobes and PCR primers, which can be used to screen for, characterizeand/or isolate a defective mammalian CXCR3 gene, which encodes areceptor having reduced or enhanced activity. Standard methods ofscreening for a defective gene can be employed, for instance. Adefective gene can be isolated and expressed in a suitable host cell forfurther assessment as described herein for mammalian CXCR3 proteins. Anumber of human diseases are associated with defects in the function ofa G-protein coupled receptor (Clapham, D. E., Cell, 75: 1237-1239(1993); Lefkowitz, R. J., Nature, 365: 603-04 (1993)).

The nucleic acids of the present invention provide reagents, such asprobes and PCR primers, which can also be used to assess expression ofreceptor (e.g., by detecting transcription of mRNA) by cells in a sample(e.g., by Northern analysis, by in situ hybridization). For example,expression in activated T lymphocytes or other cell types can beassessed.

The antibodies of the present invention have application in proceduresin which receptor can be detected on the surface of cells. The receptorprovides a marker of the leukocyte cell types in which it is expressed,particularly of activated T cells. For example, antibodies raisedagainst a receptor protein or peptide can be used to detect and/orquantify cells expressing receptor. In one embodiment, the antibodiescan be used to sort cells which express receptor from among a mixture ofcells (e.g., to isolate activated T cells, such as CD4⁺ T cells).Suitable methods for counting and/or sorting cells can be used for thispurpose (e.g., flow cytometry, fluorescence activated cell sorting).Cell counts can be used in the diagnosis of diseases or conditions inwhich an increase or decrease in leukocyte cell types (e.g., activated Tcells) is observed. The presence of an increased level of activated Tcells in a sample obtained from an individual can be indicative ofinfiltration due to an inflammatory disease or condition, such as adelayed type hypersensitivity reaction, allograft rejection, or apathologic condition, including bacterial or viral infection.

Furthermore, the antibodies can be used to detect or measure expressionof receptor. For example, antibodies of the present invention can beused to detect or measure receptor in a sample (e.g., tissues or bodyfluids from an individual such as blood, serum, leukocytes (e.g.,activated T lymphocytes), bronchoalveolar lavage fluid, saliva, bowelfluid). For example, a sample (e.g., tissue and/or fluid) can beobtained from an individual and a suitable assay can be used to assessthe presence or amount of CXCR3 protein. Suitable assays includeimmunological methods such as FACS analysis and enzyme-linkedimmunosorbent assays (ELISA), including chemiluminescence assays,radioimmunoassay, and immunohistology. Generally, a sample and antibodyof the present invention are combined under conditions suitable for theformation of an antibody-receptor complex, and the formation ofantibody-receptor complex is assessed (directly or indirectly).

The presence of an increased level of receptor reactivity in a sampleobtained from an individual can be indicative of inflammation and/orleukocyte (e.g., activated T cell) infiltration and/or accumulationassociated with an inflammatory disease or condition, such as allograftrejection, delayed type hypersensitivity reaction, or an infection suchas a viral or bacterial infection. The level of expression of amammalian CXCR3 protein or variant can also be used to correlateincreased or decreased expression of a mammalian CXCR3 protein with aparticular disease or condition, and in the diagnosis of a disease orcondition in which increased or decreased expression of a mammalianCXCR3 protein occurs (e.g., increased or decreased relative to asuitable control, such as the level of expression in a normalindividual).

Transgenic Animals

Transgenic animals, in which the genome of the animal host is alteredusing recombinant DNA techniques, can be constructed. In one embodiment,the alteration is not heritable (e.g., somatic cells, such as progenitorcells in bone marrow, are altered). In another embodiment, thealteration is heritable (the germ line is altered). Transgenic animalscan be constructed using standard techniques or other suitable methods(see e.g., Cooke. M. P. et al., Cell, 65: 281-291 (1991) regardingalteration of T lymphocytes; Hanahan, D., Science, 246: 1265-1275,(1989); Anderson et al., U.S. Pat. No. 5,399,346).

In one aspect, an endogenous mammalian CXCR3 gene can be inactivated ordisabled, in whole or in part, in a suitable animal host (e.g., by genedisruption techniques) to produce a transgenic animal. Nucleic acids ofthe present invention can be used to assess successful construction of ahost containing an inactivated or disabled CXCR3 gene (e.g., by Southernhybridization). In addition, successful construction of a hostcontaining an inactivated or disabled CXCR3 gene can be assessed bysuitable assays which monitor the function of the encoded receptor. Suchanimals can be used to assess the effect of receptor inactivation oninflammation and host defenses against cancer and pathogens (e.g., aviral pathogen).

In another embodiment, a nucleic acid encoding a mammalian CXCR3 proteinor polypeptide is introduced into a suitable host to produce atransgenic animal. In a preferred embodiment, endogenous CXCR3 receptorgenes present in the transgenic animals are inactivated (e.g.,simultaneously with introduction of the nucleic acid by homologousrecombination, which disrupts and replaces the endogenous gene). Forexample, a transgenic animal (e.g., a mouse, guinea pig, sheep) capableof expressing a nucleic acid encoding a mammalian CXCR3 receptor of adifferent mammalian species (e.g., a human CXCR3 such as the CXCR3encoded by SEQ ID NO:1) in leukocytes (such as lymphocytes (e.g.,activated T lymphocytes), natural killer cells) can be produced, andprovides a convenient animal model for assessing the function of theintroduced receptor. In addition, a test agent can be administered tothe transgenic animal, and the effect of the agent on areceptor-mediated process (e.g., inflammation) can be monitored asdescribed herein or using other suitable assays. In this manner, agentswhich inhibit or promote receptor function can be identified or assessedfor in vivo effect.

Methods of Therapy

Modulation of mammalian CXCR3 function according to the presentinvention, through the inhibition or promotion of at least one functioncharacteristic of a mammalian CXCR3 protein, provides an effective andselective way of inhibiting or promoting receptor-mediated functions. AsCXC chemokine receptors selectively expressed on activated lymphocytes,responsive to chemokines such as IP-10 and Mig whose primary targets arelymphocytes, particularly effector cells such as activated or stimulatedT lymphocytes and NK cells, mammalian CXCR3 proteins provide a targetfor selectively interfering with or promoting lymphocyte function in amammal, such as a human. Once lymphocytes are recruited to a site, otherleukocyte types, such as monocytes, may be recruited by secondarysignals. Thus, agents which inhibit or promote CXCR3 function, includingligands, inhibitors and/or promoters, such as those identified asdescribed herein, can be used to modulate leukocyte function (e.g.,leukocyte infiltration including recruitment and/or accumulation),particularly of lymphocytes, for therapeutic purposes.

In one aspect, the present invention provides a method of inhibiting orpromoting an inflammatory response in an individual in need of suchtherapy, comprising administering an agent which inhibits or promotesmammalian CXCR3 function to an individual in need of such therapy. Inone embodiment, a compound which inhibits one or more functions of amammalian CXCR3 protein (e.g., a human CXCR3) is administered to inhibit(i.e., reduce or prevent) inflammation. As a result, one or moreinflammatory processes, such as leukocyte emigration, chemotaxis,exocytosis (e.g., of enzymes) or inflammatory mediator release, isinhibited. For example, leukocytic infiltration of inflammatory sites(e.g., in a delayed-type hypersensitivity response) can be inhibitedaccording to the present method.

In another embodiment, an agent (e.g., receptor agonist) which promotesone or more functions of a mammalian CXCR3 protein (e.g., a human CXCR3)is administered to induce (trigger or enhance) an inflammatory response,such as leukocyte emigration, chemotaxis, exocytosis (e.g., of enzymes)or inflammatory mediator release, resulting in the beneficialstimulation of inflammatory processes. For example, natural killer cellscan be recruited to combat viral infections or neoplastic disease.

The term “individual” is defined herein to include animals such asmammals, including, but not limited to, primates (e.g., humans), cows,sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice orother bovine, ovine, equine, canine, feline, rodent or murine species.Diseases and conditions associated with inflammation, infection, andcancer can be treated using the method. In a preferred embodiment, thedisease or condition is one in which the actions of lymphocytes,particularly effector cells such as activated or stimulated Tlymphocytes and natural killer (NK) cells, are to be inhibited orpromoted for therapeutic (including prophylactic) purposes. In aparticularly preferred embodiment, the inflammatory disease or conditionis a T cell-mediated disease or condition.

Diseases or conditions, including chronic diseases, of humans or otherspecies which can be treated with inhibitors of CXCR3 function, include,but are not limited to:

inflammatory or allergic diseases and conditions, including systemicanaphylaxis or hypersensitivity responses, drug allergies (e.g., topenicillin, cephalosporins), insect sting allergies; inflammatory boweldiseases, such as Crohn's disease, ulcerative colitis, and ileitis;psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopicdermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g.,necrotizing, cutaneous, and hypersensitivity vasculitis);spondyloarthropathies; scleroderma; respiratory allergic diseases suchas asthma, allergic rhinitis, hypersensitivity lung diseases,hypersensitivity pneumonitis, interstitial lung diseases (ILD) (e.g.,idiopathic pulmonary fibrosis, or ILD associated with rheumatoidarthritis, or other autoimmune conditions);

autoimmune diseases, such as arthritis (e.g., rheumatoid arthritis,psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus,myasthenia gravis, juvenile onset diabetes, glomerulonephritis,autoimmune thyroiditis, Behcet's disease;

graft rejection (e.g., in transplantation), including allograftrejection or graft-versus-host disease;

other diseases or conditions in which undesirable inflammatory responsesare to be inhibited can be treated, including, but not limited to,atherosclerosis, cytokine-induced toxicity, myositis (includingpolymyositis, dermatomyositis).

Diseases or conditions of humans or other species which can be treatedwith promoters (e.g., an agonist) of CXCR3 function, include, but arenot limited to:

cancers, particularly those with leukocytic infiltration of the skin ororgans such as cutaneous T cell lymphoma (e.g., mycosis fungoides);

diseases in which angiogenesis or neovascularization plays a role,including neoplastic disease, and retinopathy (e.g., diabeticretinopathy), macular degeneration;

infectious diseases, such as bacterial infections and tuberculoidleprosy, and especially viral infections;

immunosuppression, such as that in individuals with immunodeficiencysyndromes such as AIDS, individuals undergoing radiation therapy,chemotherapy, or other therapy which causes immunosuppression;immunosuppression due congenital deficiency in receptor function orother causes. Promoters of CXR4 function can also have protectiveeffects useful to combat stem cell depletion during cancer chemotherapy(Sarris, A. H. et al., J. Exp. Med., 178: 1127-1132 (1993)).

Modes of Administration

According to the method, one or more agents can be administered to thehost by an appropriate route, either alone or in combination withanother drug. An effective amount of an agent (e.g., a receptor peptidewhich inhibits ligand binding, an anti-CXCR3 antibody or antigen-bindingfragment thereof) is administered. An effective amount is an amountsufficient to achieve.the desired therapeutic or prophylactic effect,under the conditions of administration, such as an amount sufficient forinhibition or promotion of CXCR3 receptor function, and thereby,inhibition or promotion, respectively, of a receptor-mediated process(e.g., an inflammatory response).

A variety of routes of administration are possible including, but notnecessarily limited to oral, dietary, topical, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection), andinhalation (e.g., intrabronchial, intranasal or oral inhalation,intranasal drops) routes of administration, depending on the agent anddisease or condition to be treated. For respiratory allergic diseasessuch as asthma, inhalation is a preferred mode of administration.

Formulation of an agent to be administered will vary according to theroute of administration selected (e.g., solution, emulsion, capsule). Anappropriate composition comprising the agent to be administered can beprepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils, for instance. Intravenous vehicles caninclude various additives, preservatives, or fluid, nutrient orelectrolyte replenishers and the like (See, generally, Remington'sPharmaceutical Sciences, 17th Edition, Mack Publishing Co., Pa., 1985).For inhalation, the agent can be solubilized and loaded into a suitabledispenser for administration (e.g., an atomizer, nebulizer orpressurized aerosol dispenser).

Furthermore, where the agent is a protein or peptide, the agent can beadministered via in vivo expression of the recombinant protein. In vivoexpression can be accomplished via somatic cell expression according tosuitable methods (see, e.g. U.S. Pat. No. 5,399,346). In thisembodiment, nucleic acid encoding the protein can be incorporated into aretroviral, adenoviral or other suitable vector (preferably, areplication deficient infectious vector) for delivery, or can beintroduced into a transfected or transformed host cell capable ofexpressing the protein for delivery. In the latter embodiment, the cellscan be implanted (alone or in a barrier device), injected or otherwiseintroduced in an amount effective to express the protein in atherapeutically effective amount.

EXEMPLIFICATION

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Human Chemokines

The CXC chemokines Mig, IL-8, GROα, NAP-2, GCP-2, ENA78, PF4, the CCchemokines MCP-1, MCP-2, MCP-3, MCP-4, MIP-1α, MIP-1β, RANTES, I309,eotaxin and the chemokine-related lymphotactin were chemicallysynthesized according to established protocols (Clark-Lewis, I. et al.,“Chemical synthesis, purification, and characterization of twoinflammatory proteins, neutrophil activating peptide 1 (interleukin-8)and neutrophil activating peptide 2,” Biochemistry 30: 3128-3135(1991)). The CXC chemokine IP-10 was purchased from PeproTech, RockyHill, N.J.

Example 1 Cloning of Receptor cDNA

Standard molecular biology techniques were used (Sambrook, J. et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

DNA fragments coding for putative T lymphocyte-restricted chemokinereceptors were generated using the polymerase chain reaction (PCR). Twodegenerate oligonucleotide primers were designed based on conservedmotifs of chemokine receptors. Primer design was based on the conservednucleotide sequences within transmembrane domain 2 (TM2) andtransmembrane domain 7 (TM7) of the chemokine receptors IL-8R1, IL-8R2,CC-CKR1, CC-CKR2 and the orphan receptors EBI I, LESTR, and BLR1/MDR15(EBI I, Birkenbach, M. et al., “Epstein-Barr virus-induced genes: Firstlymphocyte-specific G protein coupled peptide receptors,” J. Virol., 67:2209-2220 (1993)); LESTR, Loetscher, M. et al., “Cloning of a humanseven-transmembrane domain receptor, LESTR, that is highly expressed inleukocytes,” J. Biol. Chem. 269: 232-237 (1994); and BLR1/MDR15, Dobner,T. et al., “Differentiation-specific expression of a novel Gprotein-coupled receptor from Burkitt's lymphoma,” Eur. J. Immunol., 22:2795-2799 (1992) and Barella, L. et al., “Sequence variation of a novelheptahelical leucocyte receptor through alternative transcriptformation,” Biochem. J., 309: 773-779 (1995)).

The sequences of the primers were as follows:

SEQ ID NO:3:

5′-GGG CTG CAG CII T(T/G) (T/G) C(C/A)G AC(A/C) TIC TI(C/T) T-3′

SEQ ID NO:4:

5′-GGG TCT AGA IGG GTT IAI (G/A)CA (G/A)C(T/A) (G/A) (T/C)G-3′

(I=inosine). These primers were used in a polymerase chain reaction(PCR) to amplify DNA fragments using human genomic DNA isolated fromhuman peripheral blood lymphocytes as template as follows. A 100 μlreaction mixture containing 2 μg human genomic DNA, 1×DynaZyme buffer(Finnzymes OY, Espoo, Finland), 1.5 mM MgCl₂, 500 μM of eachdeoxynucleotide, 1 μM of both primers, and 2.5 U of DynaZyme DNApolymerase was subjected to 30 cycles (94° C. for 1 minute; 55° C. for 1minute; and 72° C. for 2 minutes) on a DNA thermal cycler (Techne PHC-2,Brouwer, Switzerland). PCR products of the predicted size (approximately700 bp) were cloned into the Gene Scribe-Z vectors pTZ18/19 U/R (USB,Cleveland, Ohio), were partially sequenced (Sanger, F. et al., “DNAsequencing with chain-terminating inhibitors,” Proc. Natl. Acad. Sci.USA, 74: 5463-5467 (1977)), and were evaluated for their similarity toknown chemokine receptors and for expression of their corresponding mRNAin leukocytes. A DNA fragment designated 2MLC22 revealed 64% nucleotidesequence identity with IL-8R2. Fragment 2MLC22 specifically hybridizedto RNA from T cells, but not monocytes or neutrophils, as assessed byNorthern blot analysis using a hybridization probe prepared byenzymatically labeling 2MLC22 with the radioactive isotope ³²P usingKlenow fragment of DNA Polymerase I and a commercially availablerandom-prime labeling kit.

Fragment 2MLC22 was enzymatically labeled with ³²P as described and usedas a probe to screen a human tetanus toxoid-specific CD4⁺ T cell (KT30)cDNA library, prepared in lambda-ZAP Express (Stratagene, Zurich,Switzerland) (Loetscher, M. et al., J. Biol. Chem ., 269: 232-237(1994)). A cDNA library was prepared in a λ ZAP Express system accordingto the manufacturer's protocol (Stratagene GMBH, Zurich, Switzerland)using poly(A)⁺ RNA from human tetanus toxoid-specific CD4⁺ T cells(KT30). The resulting library contained about 1.8×10⁶ independent cloneswith an average insert size of approximately 1.1 kb. For plaquehybridization screening, about 4×10⁵ clones were transferred ontoBiodyne nylon membranes (PALL AG, Muttenz, Switzerland) and probed with2MLC22 which had been labeled to a specific activity of 1×10⁹ dpm/μg DNAusing the high prime DNA labeling kit (Boehringer Mannheim, Mannheim,Germany). Hybridization was carried out in 50% formamide, 6×SSC, 0.5%SDS, 100 μg/ml denatured salmon sperm DNA at 42° C. for 20 hours using1×10⁶ dpm 2MLC22/ml hybridization solution. The membranes were washedonce in 2×SSC, 0.1% SDS at room temperature for 10 minutes, twice in1×SSC, 0.1% SDS at 65° C. for 30 minutes, and finally once in 0.5×SSC,0.1% SDS at 65° C. for 10 minutes. Twenty-three clones were isolatedfrom hybridization positive lambda plaques following the high stringencywashes, and the clone with the largest insert (1670 bp) was sequenced.CXCR3 cDNA was subcloned into commercially available plasmid vectors fornucleotide sequencing, generation of hybridization probes, andconstruction of stably transfected mammalian cell clones expressingCXCR3, and these CXCR3 cDNA-containing constructs are maintained in E.coli strains.

Results

A cDNA was isolated from a human CD4⁺ T cell library by searching for Tlymphocyte-specific chemokine receptors (FIG. 1, SEQ ID NO:1). This cDNAwas not recovered in the course of searching a commonly usedmonocyte-derived cDNA library or granulocyte (HL60)-derived cDNA libraryfor novel chemokine receptor cDNAs; however, a direct search of thelibraries specifically for CXCR3 cDNA has not been conducted. The CXCR3cDNA, which was shown to encode an IP-10/Mig receptor (see below), andhas an open reading frame (ORF) of 1104 bp beginning at residue 69 whichencodes a protein of 368 amino acids with a predicted molecular mass of40,659 daltons. The amino acid sequence (FIG. 2, SEQ ID NO:2) includesseven putative transmembrane segments, which are characteristic ofG-protein coupled receptors, and three potential N-glycosylation sites(Asn²², Asn³², and Asn¹⁹⁹) (FIG. 2). In addition, one threonine and nineserine residues, which are potential phosphorylation sites for receptorkinases (Palczewski, K. and J. L. Benovic, “G-protein-coupled receptorkinases,” Trends Biochem. Sci., 16: 387-391 (1991); Chuang, T. T. etal., “High expression of β-adrenergic receptor kinase in humanperipheral blood leukocytes. Isoproterenol and platelet activatingfactor can induce kinase translocation,” J. Biol. Chem., 267: 6886-6892(1992); and Giannini, E. et al., “Identification of the majorphosphorylation sites in human C5a anaphylatoxin receptor in vivo,” J.Biol. Chem., 270: 19166-19172 (1995)), can be found in the intracellularCOOH-terminal region (FIG. 2).

The 368 amino acid sequence of the receptor (IP-10/MigR, FIG. 2, SEQ IDNO:2) was aligned with the amino acid sequences of other human chemokinereceptors, including IL-8R1, IL-8R2, CC-CKR1, CC-CKR2A, CC-CKR3 andCC-CKR4. Multiple protein alignment was performed according to Higginsand Sharp (Higgins, D. G. and P. M. Sharp, “Description of the methodused in CLUSTAL,” Gene, 73: 237-244 (1988)). Double-underlined residuesin FIG. 2 represent regions of identity between IP-10/MigR and at leasttwo other chemokine receptors. Hyphens indicate gaps in the alignment.The alignment revealed several conserved motifs, particularly in thetransmembrane domains and the second intracellular loop. Significantsequence identity with CXC receptors IL-8R1 and IL-8R2, but not with theCC chemokine receptors, was observed in the third and the sixthtransmembrane domains (FIG. 2).

The sequence shares 40.9% and 40.3% amino acid identity overall with theIL-8R1 and IL-8R2 receptors, respectively, and 34.2 to 36.9% identitywith the five known CC chemokine receptors (Table 1). A lower degree ofsimilarity was found with seven-transmembrane-domain receptors that areexpressed in T cells, but which do not bind chemokines, e.g., 27.2%identity with the thrombin receptor (Vu, T.-K. H. et al., “Molecularcloning of a functional thrombin receptor reveals a novel proteolyticmechanism of receptor activation,” Cell, 64: 1057-1068 (1991)). Atruncated clone of unidentified function, with an incomplete codingsequence which can be aligned with that of FIG. 2, was previouslyisolated from a human genomic DNA library (Marchese, A. et al., “Cloningand chromosomal mapping of three novel genes, GPR9, GPR10, and GPR14encoding receptors related to interleukin 8, neuropeptide Y, andsomatostatin receptors,” Genomics, 29: 335-344 (1995)).

TABLE 1 Amino Acid Sequence Comparison of IP-10/MigR with HumanChemokine Receptors IL-8R1 IL-8R2 CC-CKR1 CC-CKR2A CC-CKR3 CC-CKR4CC-CKR5 ThrombR IP-10/MigR 40.9^(a) 40.3 34.9 34.2 34.4 35.8 36.9 27.2IL-8R1 77.1 33.7 32.9 34.3 39.7 34.3 29.1 IL-8R2 34.9 33.6 34.1 40.834.4 29.7 CC-CKR1 54.1 63.1 49.3 56.3 26.8 CC-CKR2A 50.7 46.1 68.8 24.6CC-CKR3 46.5 52.3 27.3 CC-CKR4 50.0 29.2 CC-CKR5 23.6 ^(a)Numbers referto percentage amino acid identity. Pairwise protein sequence alignmentswere carried out using the program PALIGN with an open gap cost and unitgap cost of 3 and 2, respectively.

Example 2 Biological Activity

Expression in Activated T Lymphocytes

In view of the observed chemokine selectivity, the occurrence of theIP-10/MigR in leukocytes and related cell lines was examined by Northernblot analysis. 10 μg samples of total RNA were examined from freshlyisolated human blood monocytes, neutrophils, lymphocytes (PBL),nylon-wool purified T cells, and from cultured cells including clonedhuman CD4⁺ T cells (KT30) and CD8⁺ T cells (ERCD8), cloned NK cells(ERNK57), and PBL cultured for 10 days (1-2.5×10⁶ cells/ml in RPMI 1640medium containing 2 mM glutamine, 1× non-essential amino acids, 1 mMsodium pyruvate, 100 μg/ml kanamycin, 5×10⁻⁵ M 2-mercaptoethanol, and 5%human serum) in the presence of 400 U/ml hrIL-2. (human recombinant IL-2was a gift of Dr. A. Lanzavecchia, Basel Institute of Immunology, Basel,Switzerland). Agarose gels were stained with ethidium bromide to checkthe integrity and amount of total RNA on the gel prior to blotting. RNAsamples were analyzed with ³²P-labeled 5′-fragment of the IP-10/MigR DNA(10⁹ cpm/μg DNA) at 5×10⁶ cpm/ml hybridization solution as described(Loetscher, M. et al., J. Biol. Chem., 269: 232-237 (1994)). The5′-fragment used as a Northern probe was prepared by digestion of CXCR3cDNA in pBK-CMV vector (Stratagene GMBH, Zurich, Switzerland) with PstIyielding the 724 bp 5′-end of the CXCR3 cDNA (FIG. 1).

Results

Abundant expression of mRNA of the expected size was found in the clonedCD4⁺ T cells, KT30, that were used for isolation of the receptor cDNA.Similar levels of expression were observed in the CD8⁺ T cell clone,ERCD8, and the NK cell clone, ERNK57. In contrast, in freshly isolatedblood lymphocytes and nylon-wool purified T cells, IL10/MigR transcriptswere barely detectable. However, when these cells were cultured in thepresence of IL-2, a strong upregulation was obtained, and the level ofreceptor mRNA approached that of T and NK cell clones. No IP-10/MigRtranscripts were detected under these conditions in freshly isolatedblood monocytes, neutrophil leukocytes, or eosinophil leukocytes.Additional leukocyte-related cells that did not express IP-10/MigR mRNAinclude the mast cell line, HMC-1, the promyelocytic leukemia line,HL60, the histiocytic lymphoma, U937, the chronic myelogenous leukemialine, K562, the acute T cell leukemia line, Jurkat, the acutelymphoblastic leukemia line, Molt, the B-lymphoblastic cell lines Daudiand Raji, lymphocytes from patients with chronic and acute B-lymphoidleukemia (B-CLL and B-ALL), mature basophils from a patient withbasophilic leukemia, and the erythroleukemia cell line, HEL. Bycontrast, the receptors for chemokines which have been shown previouslyto attract lymphocytes, i.e. MCP-1 MCP-2, MCP-3, MIP-1α, MIP-1β andRANTES (Loetscher, P. et al., “The monocyte chemotactic proteins, MCP-1,MCP-2 and MCP-3, are major attractants for human CD4⁺ and CD8⁺ Tlymphocytes,” FASEB J., 8: 1055-1060 (1994); Carr, M. W. et al,“Monocyte chemoattractant protein 1 acts as a T-lymphocytechemoattractant. Proc. Natl. Acad. Sci. USA 91: 3652-3656 (1994); Taub,D. D. et al., “Preferential migration of activated CD4⁺ and CD8⁺ T cellsin response to MIP-1α and MIP-1β,” Science, 260: 355-358 (1993); Schall,T. J. et al., “Human macrophage inflammatory protein α (MIP-1α) andMIP-1β chemokines attract distinct populations of lymphocytes,” J. Exp.Med., 177: 1821-1825 (1993); Schall, T. J. et al., “Selective attractionof monocytes and T lymphocytes of the memory phenotype by cytokineRANTES,” Nature, 347: 669-672 (1990)), are also found in monocytes andgranulocytes. The restricted expression of IP-10/MigR in activated Tlymphocytes and a natural killer cell line suggests that this novelreceptor can mediate selective lymphocyte recruitment.

Stable Transfectants

CXCR3 cDNA was released from pBK-CMV (Stratagene GMBH, Zurich,Switzerland) by digestion with BamHI and XbaI, and was cloned into BamHIand XbaI sites of pcDNA3 (Invitrogen BV, WB Leek, Netherlands) to yieldpcDNA3-Clone8, which is maintained and stored Escherichia coli(XL1Blue).

To generate stable transfectants, 4×10⁶ of either mouse pre-B cells(300-19) (Thelen, M. et al., FASEB. J., 2: 2702-2706 (1988)), humanpromyelocytic cells (GM-1) (Garotta, G. et al., J. Leukocyte Biol., 49:294-301 (1991)) or human acute T cell leukemia cells (Jurkat)(Loetscher, P. et al., FEBS Lett. 341: 187-192 (1994)), were transfectedby electroporation with 20 μg of receptor cDNA in pcDNA3 which waslinearized with Bgl II as described previously (Moser, B. et al.,Biochem. J., 294: 285-292 (1993)).

IP-10/MigR transfected cells were cloned by limiting dilution underG-418 (Life Technologies, Inc.) selection (1.0 mg/ml G-418 for 300-19and 0.8 mg/ml G-418 for Jurkat and GM-1 cells). G-418 resistant cloneswere screened for receptor expression by RNA Dot-blot analysis.

Ca²⁺ Flux

To determine whether the receptor was functional, clones of murine pre-Bcells (300-19), human promyelocytic cells (GM-1), and human T cellleukemia cells (Jurkat) were stably transfected with receptor cDNA asdescribed above. Activation of chemokine receptors leads to a transientrise in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_(i)), and thisassay was used to monitor signalling in the transfected cells.

Changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_(i)) weremeasured in cells loaded with fura-2 by incubation for 30 minutes at 37°C. with 0.1 nmol fura-2 acetoxymethylester per 10⁶ cells in a buffercontaining 136 mM NaCl, 4.8 mM KCl, 1 mM CaCl₂, 5 mM glucose, and 20 mMHEPES, pH 7.4. After centrifugation, loaded cells were resuspended inthe same buffer (10⁶ cells/ml), stimulated with the indicated chemokineat 37° C., and the [Ca²⁺]_(i)-related fluorescence changes were recorded(von Tscharner, V. et al., “Ion channels in human neutrophils activatedby a rise in free cytosolic calcium concentration,” Nature, 324: 69-372(1986)).

Results

A rapid [Ca²⁺]_(i) rise was observed in response to IP-10 and Mig. Thechemokine IP-10 has been shown to be expressed in cutaneous delayed-typehypersensitivity reactions (Luster, A. D. et al., “γ-Interferontranscriptionally regulates an early-response gene containing homologyto platelet proteins,” Nature, 315: 672-676 (1985); Kaplan, G. et al.,“The expression of a gamma interferon-induced protein (IP-10) in delayedimmune responses in human skin,” J. Exp. Med., 166: 1098-1108 (1987)).The chemokine designated Mig was recently identified (Farber, J. M., “Amacrophage mRNA selectively induced by gamma-interferon encodes a memberof the platelet factor 4 family of cytokines,” Proc. Natl. Acad. Sci.USA, 87: 5238-5242 (1990); Farber, J. M., “HuMIG: A new human member ofthe chemokine family of cytokines,” Biochem. Biophys. Res. Commun., 192:223-230 (1993)). Both chemokines have the CXC arrangement of the firsttwo cysteines like IL-8, but are not chemotactic for neutrophilleukocytes. It was recently reported that IP-10 attracts T lymphocytes(Luster, A. D. and P. Leder, “IP-10 a -C-X-C- chemokine, elicits apotent thymus-dependent antitumor response in vivo,” J. Exp. Med., 178:1057-1065 (1993); Taub, D. D. et al., “Recombinant humaninterferon-inducible protein 10 is a chemoattractant for human monocytesand T lymphocytes and promotes T cell adhesion to endothelial cells,” J.Exp. Med., 177: 1809-1814 (1993)), and that Mig is chemotactic fortumor-associated lymphocytes (Liao, F. et al., “Human mig chemokine:Biochemical and functional characterization,” J. Exp. Med., 182:1301-1314 (1995)).

FIGS. 3A-3C summarize the effects of IP-10 and Mig on cells transfectedwith the cDNA and expressing the functional IP-10/MigR. As shown by the[Ca²⁺]_(i) changes (FIG. 3A), the action of IP-10 and Mig wasconcentration dependent and already detectable at 1 nM, indicating thatboth chemokines have high affinity for the novel receptor. TheIP-10/MigR transfectants, by contrast, did not respond to any of 16other potential agonists at concentrations up to 100 nM, including theCXC chemokines IL-8, GROα, NAP-2, GCP-2, ENA78, PF4, the CC chemokinesMCP-1, MCP-2, MCP-3, MCP-4, MIP-1α, MIP-1β, RANTES, I309, eotaxin or thechemokine related lymphotactin (not shown). Identical results wereobtained with the murine and the human transfected cells. Theseobservations demonstrate that the novel receptor is highly selective forIP-10 and Mig. Accordingly, the receptor is referred to herein as anIP-10/Mig receptor (IP-10/MigR), or as “CXCR3”, reflecting itsspecificity for CXC chemokines.

As shown in FIG. 3B, repeated stimulation with IP-10 or Mig resulted indesensitization typical of chemokine receptors. Furthermore,cross-desensitization occurred when the cells were stimulated with IP-10followed by Mig or vice versa, confirming that the receptor has highaffinity for both chemokines. At 100 nM concentration, it became evidentthat Mig was more potent in cross-desensitization than IP-10, suggestinghigher affinity or binding stability of the IP-10/Mig receptor for Mig.

While expression of functional IP-10/MigR was demonstrated, bindingexperiments using radioactive ligands revealed non-specific bindingbetween 60 and 80% of the total, preventing determination of bindingparameters. Since IP-10 and Mig are highly cationic (pI values of 10.8and 11.1), nonspecific interaction with cell surface proteoglycans mayexplain these results. Indeed, chemokine receptor-unrelated,heparinase-sensitive binding sites for IP-10 (and PF4) have beendetected on a variety of blood and tissue cells (Luster, A. D. et al.,“The IP-10 chemokine binds to a specific cell surface heparan sulfatesite shared with platelet factor 4 and inhibits endothelial cellproliferation,” J. Exp. Med., 182: 219-231 (1995)), and heparan sulfatebinds IP-10 and Mig and prevents lymphocyte chemotaxis (not shown). Theheparin binding site is probably not involved in CXCR3 receptor binding,and inclusion of a suitable heparin derivative such as chondroitinsulfate in the reaction (e.g., in binding buffer) can be used to inhibitnon-specific binding to cells through the heparin binding site.

Chemotaxis

PBL were freshly isolated from donor blood buffy coats. Donor bloodbuffy coats were provided by the Swiss Central Laboratory BloodTransfusion Service, SRK. Isolation of buffy coat PBL was performed asdescribed in Colotta, F. et al., “Rapid killing actinomycin D-treatedtumor cells by human mononuclear cells. I. Effectors belong to themonocyte-macrophage lineage,” J. Immunol., 132: 936-944 (1984).

Freshly isolated PBL from donor blood buffy coats were used withoutfurther processing, or were used after culturing for 10 days in thepresence of IL-2 (1-2.5×10⁶ cells/ml in RPMI 1640 medium containing 2 mMglutamine, 1× non-essential amino acids, 1 mM sodium pyruvate, 100 μg/mlkanamycin, 5×10⁻⁵ M 2-mercaptoethanol, and 5% human serum in thepresence of 400 U/ml hrIL-2).

Cell migration was assessed in 48-well chambers (Neuro Probe, CabinJohn, Md., USA) using polyvinylpyrrolidone-free polycarbonate membranes(Nucleopore) with 5-μm pores for IP-10/MigR transfected cells(Loetscher, P. et al., FEBS Lett. 341: 187-192 (1994)) or with 3-μmpores for human PBL (Loetscher, P. et al., FASEB J., 8: 1055-1060(1994)). RPMI 1640 supplemented with 20 mM Hepes, pH 7.4, and 1%pasteurized plasma protein solution (Swiss Red Cross Laboratory, Bern,Switzerland) was used to dissolve the chemokines (lower wells), and todilute the cells (100,000 receptor transfectants or PBL in the upperwell). After 60 minutes at 37° C., the membrane was removed, washed onthe upper side with PBS, fixed and stained. All assays were done intriplicate, and the migrated cells were counted in five randomlyselected fields at 1,000-fold magnification. Spontaneous migration wasdetermined in the absence of chemoattractant.

Results—Transfected Cells

Transfected cells expressing the IP-10/MigR readily migrated towardIP-10 or Mig, while the non-transfected, parental cells did not respond(FIG. 3C). Both agonists showed a typically biphasic concentrationdependence. IP-10 induced migration at concentrations above 1 nM,whereas the response of Mig became detectable above 10 nM. The efficacy,which is measured by the maximum number of migrating cells, was abouttwice as high for Mig as for IP-10. These results demonstrate that theIP-10/MigR, like all known chemokine receptors in leukocytes, mediateschemotaxis in response to ligand.

Results—Human Blood Leukocytes

In agreement with the cellular distribution of the IP-10/MigR, activatedhuman T lymphocytes were found to be highly responsive to IP-10 and Mig(FIGS. 4A-4B). The activity of IP-10 and Mig as inducers of [Ca²⁺]_(i)changes (FIG. 4A) and in vitro chemotaxis (FIG. 4B) was consistent withthe effects observed using transfected cells expressing the IP-10/MigR,with IP-10 being more potent but less efficacious than Mig. Activationof the T lymphocytes by culturing in the presence of IL-2 was requiredfor induction of calcium flux and chemotaxis, and no response wasobserved with freshly isolated blood lymphocytes under the conditionsused.

Equivalents

Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

4 1670 base pairs nucleic acid double unknown CDS 69..1172 1 CCAACCACAAGCACCAAAGC AGAGGGGCAG GCAGCACACC ACCCAGCAGC CAGAGCACCA 60 GCCCAGCC ATGGTC CTT GAG GTG AGT GAC CAC CAA GTG CTA AAT GAC GCC 110 Met Val Leu GluVal Ser Asp His Gln Val Leu Asn Asp Ala 1 5 10 GAG GTT GCC GCC CTC CTGGAG AAC TTC AGC TCT TCC TAT GAC TAT GGA 158 Glu Val Ala Ala Leu Leu GluAsn Phe Ser Ser Ser Tyr Asp Tyr Gly 15 20 25 30 GAA AAC GAG AGT GAC TCGTGC TGT ACC TCC CCG CCC TGC CCA CAG GAC 206 Glu Asn Glu Ser Asp Ser CysCys Thr Ser Pro Pro Cys Pro Gln Asp 35 40 45 TTC AGC CTG AAC TTC GAC CGGGCC TTC CTG CCA GCC CTC TAC AGC CTC 254 Phe Ser Leu Asn Phe Asp Arg AlaPhe Leu Pro Ala Leu Tyr Ser Leu 50 55 60 CTC TTT CTG CTG GGG CTG CTG GGCAAC GGC GCG GTG GCA GCC GTG CTG 302 Leu Phe Leu Leu Gly Leu Leu Gly AsnGly Ala Val Ala Ala Val Leu 65 70 75 CTG AGC CGG CGG ACA GCC CTG AGC AGCACC GAC ACC TTC CTG CTC CAC 350 Leu Ser Arg Arg Thr Ala Leu Ser Ser ThrAsp Thr Phe Leu Leu His 80 85 90 CTA GCT GTA GCA GAC ACG CTG CTG GTG CTGACA CTG CCG CTC TGG GCA 398 Leu Ala Val Ala Asp Thr Leu Leu Val Leu ThrLeu Pro Leu Trp Ala 95 100 105 110 GTG GAC GCT GCC GTC CAG TGG GTC TTTGGC TCT GGC CTC TGC AAA GTG 446 Val Asp Ala Ala Val Gln Trp Val Phe GlySer Gly Leu Cys Lys Val 115 120 125 GCA GGT GCC CTC TTC AAC ATC AAC TTCTAC GCA GGA GCC CTC CTG CTG 494 Ala Gly Ala Leu Phe Asn Ile Asn Phe TyrAla Gly Ala Leu Leu Leu 130 135 140 GCC TGC ATC AGC TTT GAC CGC TAC CTGAAC ATA GTT CAT GCC ACC CAG 542 Ala Cys Ile Ser Phe Asp Arg Tyr Leu AsnIle Val His Ala Thr Gln 145 150 155 CTC TAC CGC CGG GGG CCC CCG GCC CGCGTG ACC CTC ACC TGC CTG GCT 590 Leu Tyr Arg Arg Gly Pro Pro Ala Arg ValThr Leu Thr Cys Leu Ala 160 165 170 GTC TGG GGG CTC TGC CTG CTT TTC GCCCTC CCA GAC TTC ATC TTC CTG 638 Val Trp Gly Leu Cys Leu Leu Phe Ala LeuPro Asp Phe Ile Phe Leu 175 180 185 190 TCG GCC CAC CAC GAC GAG CGC CTCAAC GCC ACC CAC TGC CAA TAC AAC 686 Ser Ala His His Asp Glu Arg Leu AsnAla Thr His Cys Gln Tyr Asn 195 200 205 TTC CCA CAG GTG GGC CGC ACG GCTCTG CGG GTG CTG CAG CTG GTG GCT 734 Phe Pro Gln Val Gly Arg Thr Ala LeuArg Val Leu Gln Leu Val Ala 210 215 220 GGC TTT CTG CTG CCC CTG CTG GTCATG GCC TAC TGC TAT GCC CAC ATC 782 Gly Phe Leu Leu Pro Leu Leu Val MetAla Tyr Cys Tyr Ala His Ile 225 230 235 CTG GCC GTG CTG CTG GTT TCC AGGGGC CAG CGG CGC CTG CGG GCC ATG 830 Leu Ala Val Leu Leu Val Ser Arg GlyGln Arg Arg Leu Arg Ala Met 240 245 250 CGG CTG GTG GTG GTG GTC GTG GTGGCC TTT GCC CTC TGC TGG ACC CCC 878 Arg Leu Val Val Val Val Val Val AlaPhe Ala Leu Cys Trp Thr Pro 255 260 265 270 TAT CAC CTG GTG GTG CTG GTGGAC ATC CTC ATG GAC CTG GGC GCT TTG 926 Tyr His Leu Val Val Leu Val AspIle Leu Met Asp Leu Gly Ala Leu 275 280 285 GCC CGC AAC TGT GGC CGA GAAAGC AGG GTA GAC GTG GCC AAG TCG GTC 974 Ala Arg Asn Cys Gly Arg Glu SerArg Val Asp Val Ala Lys Ser Val 290 295 300 ACC TCA GGC CTG GGC TAC ATGCAC TGC TGC CTC AAC CCG CTG CTC TAT 1022 Thr Ser Gly Leu Gly Tyr Met HisCys Cys Leu Asn Pro Leu Leu Tyr 305 310 315 GCC TTT GTA GGG GTC AAG TTCCGG GAG CGG ATG TGG ATG CTG CTC TTG 1070 Ala Phe Val Gly Val Lys Phe ArgGlu Arg Met Trp Met Leu Leu Leu 320 325 330 CGC CTG GGC TGC CCC AAC CAGAGA GGG CTC CAG AGG CAG CCA TCG TCT 1118 Arg Leu Gly Cys Pro Asn Gln ArgGly Leu Gln Arg Gln Pro Ser Ser 335 340 345 350 TCC CGC CGG GAT TCA TCCTGG TCT GAG ACC TCA GAG GCC TCC TAC TCG 1166 Ser Arg Arg Asp Ser Ser TrpSer Glu Thr Ser Glu Ala Ser Tyr Ser 355 360 365 GGC TTG TGAGGCCGGAATCCGGGCTC CCCTTTCGCC CACAGTCTGA CTTCCCCGCA 1222 Gly Leu TTCCAGGCTCCTCCCTCCCT CTGCCGGCTC TGGCTCTCCC CAATATCCTC GCTCCCGGGA 1282 CTCACTGGCAGCCCCAGCAC CACCAGGTCT CCCGGGAAGC CACCCTCCCA GCTCTGAGGA 1342 CTGCACCATTGCTGCTCCTT AGCTGCCAAG CCCCATCCTG CCGCCCGAGG TGGCTGCCTG 1402 GAGCCCCACTGCCCTTCTCA TTTGGAAACT AAAACTTCAT CTTCCCCAAG TGCGGGGAGT 1462 ACAAGGCATGGCGTAGAGGG TGCTGCCCCA TGAAGCCACA GCCCAGGCCT CCAGCTCAGC 1522 AGTGACTGTGGCCATGGTCC CCAAGACCTC TATATTTGCT CTTTTATTTT TATGTCTAAA 1582 ATCCTGCTTAAAACTTTTCA ATAAACAAGA TCGTCAGGAC CTTTTTTTTT TTTTTTTTTT 1642 TTTTTTTTTTTTTTTTTTTT TTTTTTTT 1670 368 amino acids amino acid linear protein 2 MetVal Leu Glu Val Ser Asp His Gln Val Leu Asn Asp Ala Glu Val 1 5 10 15Ala Ala Leu Leu Glu Asn Phe Ser Ser Ser Tyr Asp Tyr Gly Glu Asn 20 25 30Glu Ser Asp Ser Cys Cys Thr Ser Pro Pro Cys Pro Gln Asp Phe Ser 35 40 45Leu Asn Phe Asp Arg Ala Phe Leu Pro Ala Leu Tyr Ser Leu Leu Phe 50 55 60Leu Leu Gly Leu Leu Gly Asn Gly Ala Val Ala Ala Val Leu Leu Ser 65 70 7580 Arg Arg Thr Ala Leu Ser Ser Thr Asp Thr Phe Leu Leu His Leu Ala 85 9095 Val Ala Asp Thr Leu Leu Val Leu Thr Leu Pro Leu Trp Ala Val Asp 100105 110 Ala Ala Val Gln Trp Val Phe Gly Ser Gly Leu Cys Lys Val Ala Gly115 120 125 Ala Leu Phe Asn Ile Asn Phe Tyr Ala Gly Ala Leu Leu Leu AlaCys 130 135 140 Ile Ser Phe Asp Arg Tyr Leu Asn Ile Val His Ala Thr GlnLeu Tyr 145 150 155 160 Arg Arg Gly Pro Pro Ala Arg Val Thr Leu Thr CysLeu Ala Val Trp 165 170 175 Gly Leu Cys Leu Leu Phe Ala Leu Pro Asp PheIle Phe Leu Ser Ala 180 185 190 His His Asp Glu Arg Leu Asn Ala Thr HisCys Gln Tyr Asn Phe Pro 195 200 205 Gln Val Gly Arg Thr Ala Leu Arg ValLeu Gln Leu Val Ala Gly Phe 210 215 220 Leu Leu Pro Leu Leu Val Met AlaTyr Cys Tyr Ala His Ile Leu Ala 225 230 235 240 Val Leu Leu Val Ser ArgGly Gln Arg Arg Leu Arg Ala Met Arg Leu 245 250 255 Val Val Val Val ValVal Ala Phe Ala Leu Cys Trp Thr Pro Tyr His 260 265 270 Leu Val Val LeuVal Asp Ile Leu Met Asp Leu Gly Ala Leu Ala Arg 275 280 285 Asn Cys GlyArg Glu Ser Arg Val Asp Val Ala Lys Ser Val Thr Ser 290 295 300 Gly LeuGly Tyr Met His Cys Cys Leu Asn Pro Leu Leu Tyr Ala Phe 305 310 315 320Val Gly Val Lys Phe Arg Glu Arg Met Trp Met Leu Leu Leu Arg Leu 325 330335 Gly Cys Pro Asn Gln Arg Gly Leu Gln Arg Gln Pro Ser Ser Ser Arg 340345 350 Arg Asp Ser Ser Trp Ser Glu Thr Ser Glu Ala Ser Tyr Ser Gly Leu355 360 365 28 base pairs nucleic acid single unknown modified_base 11/mod_base= i modified_base 12 /mod_base= i modified_base 23 /mod_base= imodified_base 26 /mod_base= i 3 GGGCTGCAGC NNTKKCMGAC MTNCTNYT 28 27base pairs nucleic acid single unknown modified_base 10 /mod_base= imodified_base 16 /mod_base= i modified_base 18 /mod_base= i 4 GGGTCTAGANGGGTTNANRC ARCWRYG 27

We claim:
 1. An isolated nucleic acid encoding a human CXC ChemokineReceptor 3 (CXCR3) protein, wherein said CXCR3 protein binds one or morechemokines selected from the group consisting of human IP-10 and humanMig, and wherein said nucleic acid hybridizes to a second nucleic acidselected from the group consisting of the complement of SEQ ID NO:1 andthe complement of the open reading frame of SEQ ID NO:1 under highstringency wash conditions of 2×SSC, 0.1% SDS at room temperature forten minutes followed by two washes in 1×SSC, 0.1% SDS at 65° C. forthirty minutes and a final wash in 0.5×SSC, 0.1% SDS at 65° C. for tenminutes.
 2. The isolated nucleic acid of claim 1, wherein said isolatednucleic acid comprises SEQ ID NO:1 or a portion of SEQ ID NO:1comprising the open reading frame.
 3. An isolated nucleic acid encodinga protein comprising the amino acid sequence of FIG. 2 (SEQ ID NO:2). 4.An isolated nucleic acid encoding a human CXC Chemokine Receptor 3(CXCR3) protein, wherein the amino acid sequence of said CXCR3 proteinis a sequence encoded by the nucleic acid of FIG. 1 (SEQ ID NO:1).
 5. Arecombinant nucleic acid encoding a fusion protein comprising a humanCXC Chemokine Receptor 3 (CXCR3) protein, wherein said CXCR3 proteinbinds one or more chemokines selected from the group consisting of humanIP-10 and human Mig, and wherein said CXCR3 protein is encoded by anucleic acid which hybridizes to a second nucleic acid selected from thegroup consisting of the complement of SEQ ID NO:1 and the complement ofthe open reading frame of SEQ ID NO:1 under high stringency washconditions of 2×SSC, 0.1% SDS at room temperature for ten minutesfollowed by two washes in 1×SSC, 0.1% SDS at 65° C. for thirty minutesand a final wash in 0.5×SSC, 0.1% SDS at 65° C. for ten minutes.
 6. Arecombinant nucleic acid encoding a fusion protein comprising a humanCXC Chemokine Receptor 3 (CXCR3) protein, wherein the amino acidsequence of said CXCR3 protein is a sequence encoded by the nucleic acidof FIG. 1 (SEQ ID NO:1).
 7. A recombinant nucleic acid encoding a fusionprotein comprising a human CXC Chemokine Receptor 3 (CXCR3) protein,wherein the amino acid sequence of said CXCR3 protein consists of theamino acid sequence of FIG. 2 (SEQ ID NO:2).
 8. A recombinant nucleicacid construct comprising a nucleic acid encoding a human CXC ChemokineReceptor 3 (CXCR3) protein, wherein said CXCR3 protein binds one or morechemokines selected from the group consisting of human IP-10 and humanMig, and wherein said nucleic acid encoding a human CXCR3 proteinhybridizes to a second nucleic acid selected from the group consistingof the complement of SEQ ID NO:1 and the complement of the open readingframe of SEQ ID NO:1 under high stringency wash conditions of 2×SSC,0.1% SDS at room temperature for ten minutes followed by two washes in1×SSC, 0.1% SDS at 65° C. for thirty minutes and a final wash in0.5×SSC, 0.1% SDS at 65° C. for ten minutes.
 9. The recombinant nucleicacid construct of claim 8, wherein said nucleic acid encoding a humanCXCR3 protein is operably linked to an expression control sequence. 10.The recombinant nucleic acid construct of claim 8, wherein said nucleicacid encoding a human CXCR3 protein comprises SEQ ID NO:1 or a portionof SEQ ID NO:1 comprising the open reading frame.
 11. A recombinantnucleic acid construct comprising a nucleic acid encoding a human CXCChemokine Receptor 3 (CXCR3) protein, wherein the amino acid sequence ofsaid CXCR3 protein is a sequence encoded by the nucleic acid of FIG. 1(SEQ ID NO:1).
 12. A recombinant nucleic acid construct comprising anucleic acid encoding a protein comprising the amino acid sequence ofFIG. 2 (SEQ ID NO:2).
 13. A recombinant nucleic acid constructcomprising a recombinant nucleic acid of any one of claims 5, 6 and 7.14. A host cell comprising a recombinant nucleic acid encoding a humanCXC Chemokine Receptor 3 (CXCR3) protein, wherein said CXCR3 proteinbinds one or more chemokines selected from the group consisting of humanIP-10 and human Mig, wherein said nucleic acid encoding a human CXCR3protein hybridizes to a second nucleic acid selected from the groupconsisting of the complement of SEQ ID NO:1 and the complement of theopen reading frame of SEQ ID NO:1 under high stringency wash conditionsof 2×SSC, 0.1% SDS at room temperature for ten minutes followed by twowashes in 1×SSC, 0.1% SDS at 65° C. for thirty minutes and a final washin 0.5×SSC, 0.1% SDS at 65° C. for ten minutes, and wherein said hostcell is not in a transgenic animal.
 15. The host cell of claim 14,wherein said nucleic acid encoding a human CXC Chemokine Receptor 3(CXCR3) protein is operably linked to an expression control sequence.16. The host cell of claim 14, wherein said nucleic acid encoding ahuman CXC Chemokine Receptor 3 (CXCR3) protein comprises SEQ ID NO:1 ora portion of SEQ ID NO:1 comprising the open reading frame.
 17. A hostcell comprising a recombinant nucleic acid encoding a human CXCChemokine Receptor 3 (CXCR3) protein, wherein the amino acid sequence ofsaid CXCR3 protein is a sequence encoded by the nucleic acid of FIG. 1(SEQ ID NO:1), and wherein said host cell is not in a transgenic animal.18. A host cell comprising a recombinant nucleic acid encoding a proteincomprising the amino acid sequence of FIG. 2 (SEQ ID NO:2), wherein saidhost cell is not in a transgenic animal.
 19. A host cell comprising arecombinant nucleic acid encoding a fusion protein of any one of claims5, 6 and 7, wherein said host cell is not in a transgenic animal.
 20. Amethod for producing a human CXC Chemokine Receptor 3 (CXCR3) protein,wherein said protein binds one or more chemokines selected from thegroup consisting of human IP-10 and human Mig, the method comprising:(a) introducing into a host cell a nucleic acid encoding said humanCXCR3 protein, wherein said nucleic acid hybridizes to a second nucleicacid selected from the group consisting of the complement of SEQ ID NO:1and the complement of the open reading frame of SEQ ID NO:1 under highstringency wash conditions of 2×SSC, 0.1% SDS at room temperature forten minutes followed by two washes in 1×SSC, 0.1% SDS at 65° C. forthirty minutes and a final wash in 0.5×SSC, 0.1% SDS at 65° C. for tenminutes, whereby a recombinant host cell is produced having said nucleicacid operably linked to an expression control sequence; and (b)maintaining the recombinant host cell produced in step (a) underconditions whereby the nucleic acid is expressed.
 21. The method ofclaim 20, further comprising the step of isolating the human CXCR3protein.
 22. A method for producing a human CXCR3 protein comprisingmaintaining a host cell of claim 14 under conditions suitable forexpression of the recombinant nucleic acid encoding a human CXCR3protein.
 23. The method of claim 22 further comprising the step ofisolating the human CXCR3 protein.
 24. The isolated nucleic acid ofclaim 1, wherein said human CXCR3 protein comprises the extracellularN-terminal segment of the protein shown in FIG. 2 (SEQ ID NO:2).
 25. Therecombinant nucleic acid encoding a fusion protein comprising a humanCXC chemokine receptor 3 (CXCR3) protein of claim 5, wherein said humanCXCR3 protein comprises the extracellular N-terminal segment of theprotein shown in FIG. 2 (SEQ ID NO:2).
 26. The recombinant nucleic acidconstruct of claim 8, wherein said human CXCR3 protein comprises theextracellular N-terminal segment of the protein shown in FIG. 2 (SEQ IDNO:2).
 27. A method for producing a human CXC Chemokine Receptor 3protein comprising maintaining a host cell of claim 17 under conditionssuitable for expression of the recombinant nucleic acid.
 28. A methodfor producing a protein comprising the amino acid sequence of FIG. 2(SEQ ID NO:2), comprising maintaining a host cell of claim 18 underconditions suitable for expression of the recombinant nucleic acid. 29.A method for producing a fusion protein comprising a human CXC ChemokineReceptor 3 protein comprising maintaining a host cell of claim 19 underconditions suitable for expression of the recombinant nucleic acid. 30.A recombinant nucleic acid construct comprising a recombinant nucleicacid of claim
 25. 31. A host cell comprising a recombinant nucleic acidencoding a fusion protein of claim 25, wherein said host cell is not ina transgenic animal.
 32. A method for producing a fusion proteincomprising a human CXC Chemokine Receptor 3 protein comprisingmaintaining a host cell of claim 31 under conditions suitable forexpression of the recombinant nucleic acid.
 33. An isolated nucleic acidhaving a sequence that is the complement of a second nucleic acid whichencodes a human CXC Chemokine Receptor 3 (CXCR3) protein, wherein saidprotein binds one or more chemokines selected from the group consistingof human IP-10 and human Mig, and wherein said second nucleic acidhybridizes to a third nucleic acid selected from the group consisting ofthe complement of SEQ ID NO:1 and the complement of the open readingframe of SEQ ID NO:1 under high stringency wash conditions of 2×SSC,0.1% SDS at room temperature for ten minutes followed by two washes in1×SSC, 0.1% SDS at 65° C. for thirty minutes and a final wash in0.5×SSC, 0.1% SDS at 65° C. for ten minutes.
 34. An isolated nucleicacid that is the complement of SEQ ID NO:1 or the complement of aportion of SEQ ID NO:1 comprising the open reading frame.
 35. Anisolated nucleic acid that is the complement of a second nucleic acidwhich encodes a protein comprising the amino acid sequence of SEQ IDNO:2.
 36. An isolated nucleic acid that is the complement of a secondnucleic acid which encodes a human CXC Chemokine Receptor 3 (CXCR3)protein, wherein the amino acid sequence of said CXCR3 protein is asequence encoded by the nucleic acid of FIG. 1 (SEQ ID NO:1).
 37. Arecombinant nucleic acid having a sequence that is the complement of asecond nucleic acid which encodes a human CXC Chemokine Receptor 3(CXCR3) protein, wherein said protein binds one or more chemokinesselected from the group consisting of human IP-10 and human Mig, andwherein said second nucleic acid hybridizes to a third nucleic acidselected from the group consisting of the complement of SEQ ID NO:1 andthe complement of the open reading frame of SEQ ID NO:1 under highstringency wash conditions of 2×SSC, 0.1% SDS at room temperature forten minutes followed by two washes in 1×SSC, 0.1% SDS at 65° C. forthirty minutes and a final wash in 0.5×SSC, 0.1% SDS at 65° C. for tenminutes.
 38. An isolated nucleic acid encoding a human CXC ChemokineReceptor 3 (CXCR3) protein, wherein said nucleic acid comprises SEQ IDNO:1 or a portion of SEQ ID NO:1 comprising the open reading frame. 39.A host cell comprising a recombinant nucleic acid construct of claim 26,wherein said host cell is not in a transgenic animal.
 40. The isolatednucleic acid encoding a human CXCR3 protein of claim 1, wherein saidhuman CXCR3 protein induces a rapid and transient increase in theconcentration of intracellular free calcium ([Ca²⁺]_(i)) and/orchemotaxis upon chemokine binding.
 41. The recombinant nucleic acidencoding a fusion protein comprising a human CXCR3 protein of claim 5,wherein said human CXCR3 protein induces a rapid and transient increasein the concentration of intracellular free calcium ([Ca²⁺]_(i)) and/orchemotaxis upon chemokine binding.
 42. The recombinant nucleic acidconstruct comprising a nucleic acid encoding a human CXCR3 protein ofclaim 8, wherein said human CXCR3 protein induces a rapid and transientincrease in the concentration of intracellular free calcium ([Ca²⁺]_(i))and/or chemotaxis upon chemokine binding.
 43. The host cell comprising arecombinant nucleic acid encoding a human CXCR3 protein of claim 14,wherein said human CXCR3 protein induces a rapid and transient increasein the concentration of intracellular free calcium ([Ca²⁺]_(i)) and/orchemotaxis upon chemokine binding.
 44. The method for producing a humanCXCR3 protein of claim 20, wherein said human CXCR3 protein induces arapid and transient increase in the concentration of intracellular freecalcium ([Ca²⁺]_(i)) and/or chemotaxis upon chemokine binding.